Stomatal and Non-Stomatal Leaf Responses during Two Sequential Water Stress Cycles in Young Coffea canephora Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper conducted a meaningful study, and my main suggestions are as follows.

1. The meaning of the title is unclear, 'two repeated cycles of water stress' is inappropriate and needs to be reconsidered.

2. The abstract is too complex, delete the hypothesis. It needs to be rewritten in the conventional four paragraph format.

3. Too many keywords, keep at most 5-6.

4. “2.1. Micrometeorological conditions”，This part should be included in the methodology.

5.  Table 1 is not necessary to display. Or it can be placed in the attachment.

6. The significance analysis results of some of the data in Figure 4 seem to be incorrect. Please double check.

7. Overall, the figures in the paper are somewhat complicated and have poor visibility.

8. The introduction and discussion are too complicated, it is suggested to simplify them.

9. The description of the method is not clear enough. It is recommended to display it in a schematic diagram or list.

10. What is the basis for these designs? For example, the setting of various parameters. Is there any literature support or preliminary experimental results?

11. There are many abbreviations involved in the text. It is recommended to display them in a list to make the writing more concise.

12. Unofficially published literature should not be cited.

13. I don't quite understand if it's appropriate to list so many Fundings? Are these all closely related to the paper?

Author Response

Reviewer #2

The paper conducted a meaningful study, and my main suggestions are as follows.

Authors Thank you for your suggestions. We tried to modify all proposed when in accordance with journal demands. Your propositions are tagged in red.

1. The meaning of the title is unclear, 'two repeated cycles of water stress' is inappropriate and needs to be reconsidered.

                Authors: Thank you. The semantic was not correct. It was changed to:

…. two sequential water stress cycles…

2. The abstract is too complex, delete the hypothesis. It needs to be rewritten in the conventional four paragraph format.

Authors: We deleted hypotheses, despite that they are exigence of all Q1 journals in world, and substitute them with questions, that can work equally.

The ‘one paragraph’ abstract is the form demanded from the journal. So, we preserved the one paragraph form respecting MDPI demands. Please, see MDPI guides for authors and some MDPI publications, even from the proper ‘Stresses’.

3. Too many keywords, keep at most 5-6.

Authors: Journal is offering possibility of 12, but here we kept only 6, as was your recommendation: 

coffee, fluorescence; gas exchanges; memory effect; parenchyma; xylem vessel area (Line 50 in clean version).

4. “2.1. Micrometeorological conditions.This part should be included in the methodology.

Authors: The micrometeorological data were moved from the Results into Supplementary Material (actual Figure S1), because we are calling the figure about micrometeorology when we are exposing results about VPD_leaf-air (Figure 2D). If moved into M&M, it will be out of the order of figures (rule demanded by MDPI) to call Figure 8 before the Figures 3-7. So, your recommendations was respected. In the same time, the text about micrometeorological data was moved to M&M.

5. Table 1 is not necessary to display. Or it can be placed in the attachment.

Authors: We transferred this table to Supplementary Material as the Table S1.

6. The significance analysis results of some of the data in Figure 4 seem to be incorrect. Please double check.

Authors: We confirmed Figure 4 significances, and they are OK, but we change a text in actual manuscript, making it more explicit (see line 224 in the clean new text).  

7. Overall, the figures in the paper are somewhat complicated and have poor visibility.

Authors: As we are dealing with analyses of three factors, we made the highest possible simplification in presentation, using colors and the significance of factors only when significant (concept of reduced model), where the time-points were in the focus. The high-resolution figures are provided to MDPI and will help the better visibility.

8. The introduction and discussion are too complicated, it is suggested to simplify them.

Authors: Thank you. We made what was possible. Hope that you will find them useful and adequate. Reduction and various simplifications resulted in the manuscript shorter by 71 lines. Even that we added many other things as abbreviations and new diagram, as you asked. You can follow all modifications by traced copy of revision.

1. In a good rules of redaction, all presented parameters in Results, must be somehow introduced together with concept and bases for our hypotheses. To simplify the Introduction, we deleted various parts that had not been directly used in Results. Also, some movements of paragraphs were made to make the text more logical.
2. In Discussion we applied the similar strategy, and also tried to give better explanations.
3. The description of the method is not clear enough. It is recommended to display it in a schematic diagram or list.

Authors: Thank you for those suggestions.

1. We made the schematic diagram of the experiment, that helps to follow the text (terminology et al.) It is as the Figure 8 (lines 530-534 in the clean text). We really believe that it is a facilitating factor.
2. We presented all methods that are related to non-modulated and modulated chlorophyll a fluorescence, plus the measurements performed with a near infra-red leaf spectrometer, as a tables, listing the indexes, their biological, equations and references (Line 589).
3. What is the basis for these designs? For example, the setting of various parameters. Is there any literature support or preliminary experimental results?

Authors: Many ecophysiological studies are using many parameters in order to separate the most efficient ones that are responding to some of stressful conditions. Usually, we use the leaf gas exchanges (or light curve parameters) + chlorophyll fluorescence, sometimes the whole plant gas exchanges + chemical analyses + morphology. In our laboratory we have at least 40 papers of high impacts that used similar strategies to respond on questions of various scopes and treating various species. When the design of effects of three factors, they are also usual. Here a list of some of ‘doi‘ with combined settings of various ecophysiological parameters followed by three factor effects:

https://doi.org/10.1016/j.jplph.2020.153355

https://doi.org/10.1016/j.scienta.2021.110722

https://doi.org/10.3389/fpls.2023.1320552

https://doi.org/10.1111/aab.12874

https://doi.org/10.1007/s40626-023-00275-3

https://doi.org/10.1071/FP20298

https://doi.org/10.1016/j.envexpbot.2022.105051

https://doi.org/10.1111/j.1744-7348.2008.00298.x

Authors: The drought stress observed in sequential drought events is one new area of research, only few papers treated such problem, as we wrote in the manuscript. We are exploring our acquired knowledge upon the time to open new knowledge limits.

11. There are many abbreviations involved in the text. It is recommended to display them in a list to make the writing more concise.

Authors: Thank you. We made a list and put it before the Introduction. They are in lines 51-60 of the clean text.

12. Unofficially published literature should not be cited.

Authors: Thank you. We deleted what is still in revision. One manuscript was accepted on 11^th of June, and those days I assigned (in behave ff all authors) the license for publishing. We do not know only the number, but it will be re4solved. Cambridge site is in problems because of hacked attack, and everything is delayed for 2-3 months, but we will have the e-number (or DOI) those days.

13. I don't quite understand if it's appropriate to list so many Fundings? Are these all closely related to the paper?

Authors: Yes, they are all appropriate and necessary. As we examined stomatal (leaf gas exchanges) and not stomatal responses (fluorescence, reflectance and anatomy) to two subsequent drought cycles, we tried to respond which approach/methodology would be the most efficient in detection of stresses. During the execution, we have 17 automatic responses of non-modulated fluorescence, and among them we chose only four. We had much more data and reduced them to those presented in manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

An exceptionally well-prepared paper!

It is highly topical and extremely relevant against the backdrop of the climatic challenges of climate change for coffee cultivation – especially due to severe droughts and their impact on the yield situation for Canephora.

The experimental design and implementation are well-structured and very coherent.

I can only provide a few questions or constructive suggestions for the smallest possible improvements:

The pH value of the potting soil used, as well as the exact water retention capacity, would haven been interesting to know and should be specified. The pH value in particular is an essential control parameter for the possible uptake of nutrients by the plant.

Is there any information on the microbiome in the soil? The uptake of precursors by microorganisms from the anthocyanin and carotenoid groups is likely to be of great importance and interest in this context. 

It would also have been nice if, in addition to the two Robusta varieties of Canephora, plants of the Conillon variety (as one of the most widespread Canephora varieties in Brazil) had also been considered in the study.

Once again, congratulations on this truly exceptional paper.

Comments for author File: Comments.pdf

Author Response

Reviewer #3

An exceptionally well-prepared paper!

                Authors: Thank you for your kindness.

It is highly topical and extremely relevant against the backdrop of the climatic challenges of climate change for coffee cultivation – especially due to severe droughts and their impact on the yield situation for Canephora.

The experimental design and implementation are well-structured and very coherent.

                Authors: Thank you for your kindness.

I can only provide a few questions or constructive suggestions for the smallest possible improvements:

The pH value of the potting soil used, as well as the exact water retention capacity, would have been interesting to know and should be specified. The pH value in particular is an essential control parameter for the possible uptake of nutrients by the plant.

Authors: We added the Table S2 in Supplementary Material and the following text about the soil composition before the execution of experiment (lines 503-507 of clean new version):

Soil pH determined in H₂O was 5.6, effective cation exchange capacity = 4.0 cmol_c dm^-3, , basis saturation 78.3 % (Table S2). The concentration of P, K, Fe, Cu, Zn, Mn, S and B was 25.5, 372, 25.5, 0.4, 6.4, 27.6, 429.4 and 6.8 mg dm-³, respectively, while of Ca, Mg, Al, H+Al and Na was 1.1, 1.7, 0.0, 1.1, 0.2 cmol_c dm-³, respectively. The C and organic matter concentrations were 0.6 and 1 %.

 

Is there any information on the microbiome in the soil? The uptake of precursors by microorganisms from the anthocyanin and carotenoid groups is likely to be of great importance and interest in this context.

Authors: That’s true, but we do not have any information. Hope that soil chemical analyses will be sufficient.

It would also have been nice if, in addition to the two Robusta varieties of Canephora, plants of the Conillon variety (as one of the most widespread Canephora varieties in Brazil) had also been considered in the study.

Authors: That’s true, and in one new experiment with the whole plant photosynthesis, we are considering some options.

Once again, congratulations on this truly exceptional paper.

                Authors: Thank you for your kindness.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has made extensive revisions to the manuscript and addressed my concerns. It is now ready for publication.

Author Response

The author has made extensive revisions to the manuscript and addressed my concerns. It is now ready for publication.

 

 Authors: Thank you for your positive evaluation of the revised manuscript.