Identifying Grapevine Rootstocks Tolerant to Copper Excess

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

2.6 Statistical analysis

Yes it is correct, in My opinion the interaction between factors would be more informative (the effect of mutual influence) , but You  prefer to accent differences within factors OK

Author Response

We understand the reviewer's suggestion. An analysis was performed with the interaction between the factors. And when the analysis was significant, the analysis was unfolded.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The current study aimed to identify grapevine rootstocks (i.e., Paulsen 1103, IAC 572, SO4, and Isabel) with the potential to tolerate excessive Cu concentrations (i.e., 0.3 µM Cu and 80 µM Cu). The results indicated that Paulsen 1103 was the most tolerant to Cu excess, whereas Isabel was the most sensitive. 

The text requires major revisions of the English language.

In the text, there are numerous parts highlighted in yellow by the authors.

Some parts in the introduction are useless to the text

Lines 50-56 to delate

Shoot height was measured at the end of the experiment with the aid of measuring tape. Why only at the end?

Chlorophyll a fluorescence analysis and net P were carried out 15 days after treatment application. Why only at this time?

Authors should specify the repetitions employed

How do the authors explain a significant increase in copper in control shoots (Isabel)?

How do the authors explain the higher WUE values ​​in high doses of copper?

Figures 3a, 3b, 2c are superfluous

Comments on the Quality of English Language

Extensive editing of English language required

Author Response

Shoot height was measured at the end of the experiment with the aid of measuring tape. Why only at the end?

Reply: we measured plants only at the end of the experiment. Indeed measuring at the beginning and during the course of the experiment would also be informative, allowing us to use relative growth and growth rates. However, since we are comparing our treated plants with controls that were cultivated side-by-side, we do not think that conclusions would change in any significant way – i.e., we can tell which genotypes grow more without Cu, as well as what is the effect of Cu treatment. Therefore, although certainly informative and more detailed, these extra measurements would not affect our conclusions.

Chlorophyll a fluorescence analysis and net P were carried out 15 days after treatment application. Why only at this time?
Reply: we believe a similar explanation as above applies here. Measuring photosynthetic parameters at earlier or later time points could help in comparisons as how Cu affects each genotype in time (i.e., when effects are observed), but would not change significantly our conclusions. Since we have control plants, we can directly compare treatment and genotype effects. We chose 15 days because at this point we expected effects to be observable, based on how the plants show other toxicity symptoms, which turned out to be the case.

 

Authors should specify the repetitions employed

Reply: all experiments were conducted with four replicates, as described in Methods. All analyses were performed using these independent replicates. The information was not fully mentioned in all described analyses

How do the authors explain a significant increase in copper in control shoots (Isabel)?
Reply: our data does not allow us to explain fully this observation, only to speculate. One possibility is that Isabel, being a genotype that is usually used as a scion, has a larger Cu root to shoot translocation because of genetic factors, such as low root vacuolar sequestration of Cu. Upon exposure to Cu, Isabel could be retaining Cu in roots as other genotypes, but also decreasing shoot growth, which could in turn affect Cu accumulation in shoots.. More importantly, it is key to notice that the genotypes tested do not vary Cu concentration to a large extent in shoots (some are statistically significant, but of low effect size), which suggest that most effects are secondary to Cu accumulation in roots. For sure, in order to explain why Isabel behave differently, we would need to perform experiments focused on mechanisms and genes that are playing a role in Cu homeostasis, which is beyond the scope of this study.

How do the authors explain the higher WUE values in high doses of copper?
Reply: water use efficiency is a ratio of Net Photosynthesis and Transpiration Rate. As it can be seem in Fig 2a and 2d, while both decrease in all genotypes under Cu excess, Transpiration rate decreases to a larger extent. Therefore, the ratio becomes positive, since Net Photosynthesis decreases not as much. However, it is important to interpret the ratio together with the other two values: although WUE increases under Cu excess, that means less water per carbon fixed is used, but the total amount of carbon fixed is lower compared to the same genotype under control conditions.

Figures 3a, 3b, 2c are superfluous

Reply: We understand the reviewer suggestion, and agree that Figures 3a and 3b are not necessary. We would rather keep Figure 2c, since it allows interpretation of IRGA measurements together with the other parameters. We hope that is OK for the reviewer.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This study aimed to assess the tolerance of each genotype to copper stress by physiological comparison of four grape rootstocks under different concentration treatments and is ready for publication with modifications. However, some doubts exist and are suggested below:

1. Materials and Methods

(1) The description of the aeration device under hydroponic conditions is missing from the material methods and needs to be added;

(2) Why was 15d chosen as the time of sampling and on what basis? Suggestions can be reflected in the relevant research progress in the introduction section;

(3)What is the rationale for selecting 80 µM as the high copper concentration? Suggestions can be reflected in the relevant research progress in the introduction section;

2. Statistical analysis

(1) Lines 162-164 in the data analysis are suggested to be placed in 2.1 Test Materials and Growth Conditions;

(2)Whether a variance chi-square test was indicated prior to data processing in rows 165-169 of the data analysis;

3. Other

The units for stomatal conductance Gs in Figure 2b are incorrect and should read: mmol·m^-2·s^-1.

4. Conclusion

The conclusion of the article feels a bit lengthy; make a direct conclusion and delete unnecessary explanations and narratives.

Author Response

1. Materials and Methods

(1) The description of the aeration device under hydroponic conditions is missing from the material methods and needs to be added;

Reply: Thank you for noticing. We added that to the text.

(2) Why was 15d chosen as the time of sampling and on what basis? Suggestions can be reflected in the relevant research progress in the introduction section;

Reply: Measuring photosynthetic parameters at earlier or later time points could help in comparisons as how Cu affects each genotype in time (i.e., when effects are observed), but would not change significantly our conclusions. Since we have control plants, we can directly compare treatment and genotype effects. We chose 15 days because at this point we expected effects to be observable, based on how the plants show other toxicity symptoms, which turned out to be the case.

(3) What is the rationale for selecting 80 µM as the high copper concentration? Suggestions can be reflected in the relevant research progress in the introduction section;

Reply: the concentration used (80µM) was based on previous studies by our group (Trentin et al., 2023).

 Trentin, E.; Ferreira, P. A. A.; Ricachenevsky, F. K.; Facco, D. B.; Hammerschmitt, R. K.; Morsch, L.; Tarouco, C. P.; Nicoloso, F. T.; Araujo, M. M.; Berghetti, A. L. P.; de Melo, G.W. B.; Brunetto, G. Growth, biochemical and physiological response of grapevine rootstocks to copper excess in nutrient solution, South African Journal of Botany, v. 162, p. 360-369, 2023.

2. Statistical analysis

(2) Whether a variance chi-square test was indicated prior to data processing in rows 165-169 of the data analysis;

Reply: We did not perform this analysis. Instead, we performed normality test and homocedasticity test before ANOVA. These information were added to the methods.

3. Other

The units for stomatal conductance Gs in Figure 2b are incorrect and should read: mmol·m^-2·s^-1.

Reply: thank you for noticing. We changed the figure.

 

4. Conclusion

The conclusion of the article feels a bit lengthy; make a direct conclusion and delete unnecessary explanations and narratives.

Reply: thank you. We changed following reviewers suggestions.

 

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

In this manuscript, the authors identified the tolerance of four grapevine rootstock genotypes to excessive Cu concentration. Combined with various physiological and biochemical parameters, the different responses of these rootstocks to Cu excess were analyzed, and it was identified that Paulsen 1103 was the most tolerant to Cu excess, while Isabel was the most sensitive. These results provide a basis for understanding the mechanisms of Cu tolerance in grapevine rootstocks. Nevertheless, some experimental and writing details require careful explanation and revision by the authors. I hope these comments are helpful to improve the manuscript.

 

1. The authors said Paulsen1103 was the most tolerant to Cu excess, whereas Isabel was the most sensitive. In Figure 1, compared with the control treatment, high Cu treatment significantly affected the growth of IAC572 rootstock, rather than Isabel. Please explain these data.

2. In Table 2, only the changes of micronutrient concentration in the roots of grapevine rootstocks were described in the manuscript, and the shoot was not described. Please supplement.

3. Please add a scale bar in Figure S1, S2.

4. Cu treatment significantly affected the Fe and Mn concentrations in rootstocks, potential relationships between which can be described in the discussion.

5. There are several writing errors: Line 97: CuSO4.5H2O; H2O2 (Line178, Line 335, Line 337, Line 344, Line 350); CO2 (Line 362, Line 370, Line 377); m-2 s-1 (Line 314-320).

Author Response

1. The authors said Paulsen1103 was the most tolerant to Cu excess, whereas Isabel was the most sensitive. In Figure 1, compared with the control treatment, high Cu treatment significantly affected the growth of IAC572 rootstock, rather than Isabel. Please explain these data.

Reply: indeed, IAC is vigorous, and showed a marker decrease in shoot growth when exposed to Cu excess. However, root growth was increase, suggesting that the rootstock genotype changed carbon partition towards root growth, which might be important for Cu excess response. Isabel, on the other hand, considering visual phenotype as well as other measurements, is overall more sensitive to Cu excess.

In Table 2, only the changes of micronutrient concentration in the roots of grapevine rootstocks were described in the manuscript, and the shoot was not described. Please supplement.

Reply: Thank you. We added that description to the results.

3. Please add a scale bar in Figure S1, S2.

Reply: We understand the reviewer's suggestion, but at the time we took the photo we did not include any material with reference to the scale.

4. Cu treatment significantly affected the Fe and Mn concentrations in rootstocks, potential relationships between which can be described in the discussion.

Reply: we added new information to the discussion.

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Accept in present form

Comments on the Quality of English Language

Moderate editing of English language required.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Paper is interestig especially for vineyards where copper as Bordeaux mixture or any other copper compounds are used since many year results of Cu accumulation in soil, so response of vine cultivars to copper excess is important.

Some comments:

Line 149-150 ; how much part of root and shoots were frozen

Line 155 L: 1 ml K-1 mol L-1 what does it mean, concentration?

2.6 Statistical analysis

TWO WAY ANOVA - and then Tukey test is in my opinion enough to create/separate homogenous groups for all data and additional comparison for control and high Cu is no needed, the interaction varieties (rootstocks) x copper dose for analysed parameters can be presented as bars or in tables only with letters denoting homogenous grups . Asterisk and letter mix makes figures and tables less clear, for example Fig 1 a letter b for Paulsen 1103, SO4, Isabel means not significant difference? but b and astersk for SO4 significant?. But this is up for discussion.

3.1 Growth of different grapevine rootstock genotypes

In my opinion if statistical evaluation exists, so it should be used in coments to results, beside "reduction by 41, 35 and 13% " of height the comment have to contain that these varieties significantly differ from control, and so on for other biometrical data

3.2 Nutrient homeostasis

as above comment needs contain significance or not between examined objects

and and also refers to the remaining paragraphs

for example fig could look like, but this is up for discussion

 

Author Response

Reviewer #1:

Paper is interesting especially for vineyards where copper as Bordeaux mixture or any other copper compounds are used since many year results of Cu accumulation in soil, so response of vine cultivars to copper excess is important.

Some comments:

Line 149-150; how much part of root and shoots were frozen.

Reply: we did not weight the bulk amount of tissue that was collected and frozen at the end of experiments. However, all protocols used afterwards based on weight were performed based on defined amounts of sample. Given the total used, we had at least 2 g of tissue collected per biological replicate.  

Nós não pesamos as quantidades coletadas. Mas foram coletadas amostras para ter no mínimo 2g de matéria fresca de tecido, necessário para as análises.

 

Line 155 L: 1 ml K-1 mol L-1 what does it mean, concentration?

Reply:Thank you for noticing. We corrected in the text, which now reads as “0.5 ml of potassium phosphate buffer (10 mM) (pH 7.0) and 1 ml of KI (1M)”

A correção foi realizada.

 

2.6 Statistical analysis

TWO WAY ANOVA - and then Tukey test is in my opinion enough to create/separate homogenous groups for all data and additional comparison for control and high Cu is no needed, the interaction varieties (rootstocks) x copper dose for analysed parameters can be presented as bars or in tables only with letters denoting homogenous groups. Asterisk and letter mix makes figures and tables less clear, for example Fig 1 a letter b for Paulsen 1103, SO4, Isabel means not significant difference? but b and astersk for SO4 significant? But this is up for discussion.

Reply: Our study was conducted in as a factorial 4 X 2, with the goal of evaluating the behaviour of each rootstock under control and high Cu concentrations. We used comparisons of genotypes within the same treatment (control or high Cu), and of comparing genotypes-specific response to treatment. Other studies published in high-impact journals used similar statistical analyses. Here are some examples: 

Effects of Rhizophagus clarus and P availability in the tolerance and physiological response of Mucuna cinereum to Copper. https://doi.org/10.1016/j.plaphy.2017.11.006

Rhizophagus clarus and phosphate alter the physiological responses of Crotalaria juncea cultivated in soil with a high Cu level. https://doi.org/10.1016/j.apsoil.2015.02.008

Potential of vermicompost and limestone in reducing copper toxicity in young grapevines grown in Cu-contaminated vineyard soil.

 https://doi.org/10.1016/j.chemosphere.2019.03.141

Effect of dynamic controlled atmosphere on volatile compound profile and quality of pears. https://doi.org/10.1016/j.scienta.2024.112910

We would like to maintain the statistical analyses as is, since it is not wrong, and it would mean changing all figures, as well as not using the same standard we and other use. Please let us know if that’s adequate.

 

 

3.1 Growth of different grapevine rootstock genotypes

In my opinion if statistical evaluation exists, so it should be used in coments to results, beside "reduction by 41, 35 and 13% " of height the comment have to contain that these varieties significantly differ from control, and so on for other biometrical data

Reply: We are only mentioning percentage differences when these differences are statistically significant,. We believe that was implicit in the results description, but we made it more explicit. Information were added to text, as requested.

 3.2 Nutrient homeostasis

as above comment needs contain significance or not between examined objects and also refers to the remaining paragraphs for example fig could look like, but this is up for discussion.

Reply: We Thank you for the comment. As mentioned in item 2.6, our study was conducted in a 4X2 factorial. In this example, the experimental design would be 3 X 3 +1, in which we would have three Se doses and three Zn doses, besides the control treatment. Author performed the analysis using ANOVA with one factor to test differences between averages in three or more independent groups. When the study involved one or more factors, we have a multiple ANOVA. In an ANOVA in which all combinations are considered is a factorial ANOVA. In studies with more than one factor, this approach allow to analyze the isolated effects of factors and their interactions. When the interaction between factors is significant, a strategy for analyses could be the untangling of degrees of freedom of one factor within each level of another factor. Similar to what we used in our study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewer’ comments:  

This article selects four types of grape rootstocks for the identification and evaluation of copper tolerance. Growth, nutrient concentration in tissue, physiological and biochemical parameters were assessed. The results indicate that Paulsen 1103 is the most tolerant to Cu excess, whereas Isabel is the most sensitive.

The author did not conduct in-depth analysis of cell structure and transcription levels, but only judged whether the four rootstocks were copper tolerant based on physiological indicators, which was slightly decisive.

Here listed some advises that needs to be addressed:

(1)   “Isabel presented the greatest sensitivity to Cu excess, this outcome was clearly observed through leaf wilting and yellowing”. The authors should provide pictures.

(2) The measurement unit is inconsistent throughout the manuscript and the writing is not standardized. For example, in 2.3 “1,000 μmol m2 s-1”, in 3.4 “1,500 nmolm-2s-1”, etc.

(3) The molecular formula of chemical substances is written improperly, such as “H2O2”, “CO2”, etc. In addition, there are some writing errors in the article, such as, “Funding: Please add:”; “Maximum electron transport rate (ETRmax) was determined in the dark period - before dawn (5:00-6:00 am) (Ferreira et al., 2015)”.

(4) The Discussion section needs to be rewritten. In this section, author should tell reader what was your new finding in this study when compared to previous studies and then discussed the possible mechanism or scientific value of the new finding. From the present Discussion, the authors could not properly and concisely state the main point of this study.

 

 

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Reviewer #2:

Comments and Suggestions for Authors

Reviewer’ comments: 

This article selects four types of grape rootstocks for the identification and evaluation of copper tolerance. Growth, nutrient concentration in tissue, physiological and biochemical parameters were assessed. The results indicate that Paulsen 1103 is the most tolerant to Cu excess, whereas Isabel is the most sensitive.

The author did not conduct in-depth analysis of cell structure and transcription levels, but only judged whether the four rootstocks were copper tolerant based on physiological indicators, which was slightly decisive.

Here listed some advises that needs to be addressed:

(1) “Isabel presented the greatest sensitivity to Cu excess, this outcome was clearly observed through leaf wilting and yellowing”. The authors should provide pictures.

Reply: We are adding the images as requested.

 

(2) The measurement unit is inconsistent throughout the manuscript and the writing is not standardized. For example, in 2.3 “1,000 μmol m2 s-1”, in 3.4 “1,500 nmolm-2s-1”, etc.

Reply: Thank you. Corrections were made considering the reviewer’s suggestions.

 

 

(3) The molecular formula of chemical substances is written improperly, such as “H2O2”, “CO2”, etc. In addition, there are some writing errors in the article, such as, “Funding: Please add:”; “Maximum electron transport rate (ETRmax) was determined in the dark period - before dawn (5:00-6:00 am) (Ferreira et al., 2015)”.

Reply: Thank you. The changes were added to the text.

 

(4) The Discussion section needs to be rewritten. In this section, author should tell reader what was your new finding in this study when compared to previous studies and then discussed the possible mechanism or scientific value of the new finding. From the present Discussion, the authors could not properly and concisely state the main point of this study.

Reply: Thank you for your comment. We have worked on the discussion to improve it. We also worked on the conclusion to make it clearer and more concise.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you very much for your reply, but some of the review comments made last time remain unchanged. In addition, there are still some questions that need to be improved.

1.     The molecular formula of chemical substances is written improperly, such as “H2O2”, “CO2”, etc.

2.     I suggest to attach the images of chlorophyll fluorescence, which will clearer the response of the four rootstocks to resistance to Cu stress.

3.     Paulsen 1103 is the most tolerant genotype, BUT why it also had the highest H₂O₂ accumulation under high Cu? Generally, the resistant varieties are also more capable of scavenging reactive oxygen species, please discuss it.

 

4.     The formatting of the writing and citation of references in the article is very poorly organized.

Comments on the Quality of English Language

Moderate editing of English language required.

Author Response

To the Editor and Reviewers,

The authors thank you for contributing your knowledge and time to improve our manuscript. All altered text in the manuscript are colored red.

1. The molecular formula of chemical substances is written improperly, such as “H2O2”, “CO2”, etc.

Reply: The changes were made.

2. I suggest to attach the images of chlorophyll fluorescence, which will clearer the response of the four rootstocks to resistance to Cu stress.

Reply: We appreciate the suggestion. Unfortunately, at the time of analysis, we do not record the evaluations with photographs.

3. Paulsen 1103 is the most tolerant genotype, BUT why it also had the highest H2O2 accumulation under high Cu? Generally, the resistant varieties are also more capable of scavenging reactive oxygen species, please discuss it.

Reply: Indeed, that's interesting and counterintuitive. It is obviously expected to find tolerant genotypes with lower hydrogen peroxide and higher oxidative metabolism enzymes activity. However, it is important to remember that hydrogen peroxide (and the oxidative metabolism as a whole) do not function always neatly as expected. Hydrogen peroxide is a signaling molecule and might be used by a tolerant cultivar to better signal stress, which might be related to tolerance. Of course, we cannot make assumptions on the reasons why Paulsen does show the highest hydrogen peroxide levels. Still, it's important that the maximum values observed are low compared to other reported values in grapevine tissues even under control conditions (10.3389/fenvs.2015.00020 and 10.1016/j.plaphy.2018.07.036) as well as in pear and peach (10.7717/peerj.14947). Therefore, we cannot rule out a signaling role for hydrogen peroxide at the concentration found in Paulsen tissues. 

It is also important to note that, at least in leaves, SOD activity (which generates hydrogen peroxide inside the cells) is also quite high, at least in Paulsen shoots. IN roots, the high values could be high due to hydrogen peroxide from other sources, such as peroxidases. However, we can only speculate on that, with the data at hand. Still, this is an interesting observation that deserves more attention in the future.

4. The formatting of the writing and citation of references in the article is very poorly organized.

Reply: The changes were made.