Metabolic, Nutritional and Morphophysiological Behavior of Eucalypt Genotypes Differing in Dieback Resistance in Field When Submitted to PEG-Induced Water Deficit

Round 1

Reviewer 1 Report

The paper written by Madeira et al. is entitled Water deficit induces different metabolic, nutritional and morphophysiological behaviour in Eucalypt genotypes exhibiting different resistance level to dieback. Looking at the title, a reader would expect to find results of different genotypes under different water stress conditions. The article is interesting, novel and well written. However, the authors make a serious methodological error.

 

In all the experiments carried out, except for the biochemical analyses, the results are grouped by treatment or genotype. This leads to several problems. In the case of grouping all the results by genotype, when we obtain the mean of a parameter, this data is the result of three different treatments, i.e. control, 100PEG and 300 PEG. In the second case, when analysing the treatments, the four genotypes are grouped in each treatment. Undoubtedly, neither of the two analyses allows us to obtain answers to the objective of the work.

 

The assumption you get when you read the title of the paper is that there are different genotypes that respond differently to various water deficit conditions. However, this is not observed in the results. For example, if we look at the root dry matter results, there are no significant differences between genotypes. The general hypothesis would be that the resistant genotype has a difference in root dry matter with the sensitive species. For this we should break down the results by genotype and by treatment, not group them together; by doing the latter, the variability is being lost and is being absorbed by the large number of samples.

 

When we analyse the results of the susceptible genotype, we observe that it has less leaf dry matter and a smaller diameter. This is to be expected, as it is more susceptible to water deficit and must regulate water loss; however, it is the genotype with the highest photosynthetic rates. How can it show higher photosynthetic rates and, on the other hand, lower biomass? It could be that the control treatment introduces noise into the analysis by increasing the mean photosynthetic values. Therefore, in this case, and as previously mentioned, the results of each genotype in each water deficit situation should be separated. In this way, it will be possible to appreciate the real differences between the genotypes depending on the treatment.

 

With regard to the second case, grouping the genotypes to observe how they respond to the treatments, we make the same mistake again. This analysis of the data does not provide any meaning. In fact, in almost all the figures comparing treatments, there are no significant differences. This is obviously an error due to the focus of the results.

In addition to the above, when you read the keywords, one of them is osmotic stress. If you look at the material and methods, the heading indicates water potential, and in the results, figure 1 also represents water potential. The authors cannot talk about water potential as they have not measured it. Instead, they have measured the osmotic potential. This also leads to a problem, as there does not have to be a direct relationship between the osmotic potential and the water potential on the plant. Even if there is a lower osmotic potential in the soil, the regulation of the cellular water status by the plant is done through transpiration and compatible osmolytes. This can lead to a substantial change in water uptake, and consequently to significant variations in soil water potential. Therefore, figure 1 and any allusion to water potential should be changed to osmotic potential.

In summary, the paper has potential but should be rewritten. The figures in which the treatments are grouped together should be removed, and instead the results of each genotype should be analysed according to the water deficit treatments. Obviously hedgerows means changing the discussion. The approach to osmotic potential should also be rewritten.

Author Response

The paper written by Madeira et al. is entitled Water deficit induces different metabolic, nutritional and morphophysiological behaviour in Eucalypt genotypes exhibiting different resistance level to dieback. Looking at the title, a reader would expect to find results of different genotypes under different water stress conditions. The article is interesting, novel and well written. However, the authors make a serious methodological error.

 

In all the experiments carried out, except for the biochemical analyses, the results are grouped by treatment or genotype. This leads to several problems. In the case of grouping all the results by genotype, when we obtain the mean of a parameter, this data is the result of three different treatments, i.e. control, 100PEG and 300 PEG. In the second case, when analysing the treatments, the four genotypes are grouped in each treatment. Undoubtedly, neither of the two analyses allows us to obtain answers to the objective of the work.

In this report, we performed one experiment from which plants were sampled for different analysis.

The analysis was carried out, as previously observed by reviewer 1, grouped by treatment and genotype because most of the characteristics evaluated did not show significant interaction, except for sulphur, cupper, and total amino acids. Sulphur and cupper were further analyzed in separate while amino acid content was not. This is due the former two variables contributed to the principal component analysis, and the latter did not. We amended our discussion trying to correct the errors in the original manuscript version. Although we are sending a reviewed version, we reinforce our request for an extension of the deadline to fulfill the corrections that were requested of the ones that will be observed in the present file.

Despite our discussion was performed based on the empirical data from the forestry company, we simulated water deficit and evaluated the genotypes (eucalyptus clones) according their responses to water deficit treatments. The grouping according genotypes and treatments were accomplished as reviewer 1 already pointed out, and there was no interaction between genotype and treatment, for the genotypes that were used in this experiment.

Except for amino acids content, sulphur and copper, these results are the response outcomes of eucalypt genotypes submitted to water deficit treatments that can be extrapolated to eucalypt genotypes in general. The same interpretation can be applied to the water treatments (control and deficit). Accordingly, we can conclude a general behavior for clones and treatments, based on the sample constituted of 4 commercial genotypes. It is different from saying that we will not have a differential (significant interaction) when evaluating different and more genotypes or clones, as we observed in Corrêa et al (2023).

Evaluating other variables and a greater number of genotypes the expected differences were highlighted (Corrêa et al 2023), what reinforce a cohort senescence theory contextualized for water deficit and EPD resistance (Picoli et al 2021, Mueller-Dombois 1986), and the existence of several strategies contributing to the tolerance phenotype (Picoli et al 2021).

Please, observe that the objectives prior and originally mentioned in the abstract “Aiming at the early identification of tolerant genotypes, we evaluated plantlets of four commercial clones with divergent behavior in field conditions” and at the end of the introduction “Thus, this work aimed to characterize the morphological, physiological, nutritional and metabolic responses of seedlings of commercial eucalypt genotypes, with different levels of tolerance to dieback, submitted to osmotic stress.” are fulfilled. We accomplished the identification of correspondent genotype that is more tolerant and characterized general morphological, physiological, nutritional and metabolic responses associated with a general response of each genotype and water deficit treatment.

In the present experiment, we were guided by empirical data collected in field conditions and used a restricted number of genotypes. This can also contribute the results with no significant interaction (genotype x treatment). This was an initial approach aiming the incorporation of metabolic traits into the evaluation process.

With the latter report (Corrêa et al 2023), we became aware that SuzT behavior more similar to the semi-tolerant clone according our evaluation. This information can possibly justify the lack of interaction between genotype and treatment, as were observed in the ulterior experiment. We hope, in future experiments, to test a workable number of markers (characteristics) and genotypes, further testing this approach.

 

The assumption you get when you read the title of the paper is that there are different genotypes that respond differently to various water deficit conditions.

The reviewer 1 has a point and is correct. It was amended. Please, observe the modification in the title and throughout the manuscript.

 

However, this is not observed in the results. For example, if we look at the root dry matter results, there are no significant differences between genotypes. The general hypothesis would be that the resistant genotype has a difference in root dry matter with the sensitive species. For this we should break down the results by genotype and by treatment, not group them together; by doing the latter, the variability is being lost and is being absorbed by the large number of samples.

Our experiment and results will due for general trends towards the resistant phenotype. Despite we were eager to evaluate metabolome traits, we were, forcefully, restricted by the number of genotypes approached in this experiment, what impacted, negatively our results.

Since we had no interaction and, considerable amount of the traits did not show significant differences, the break down of the results by genotype and treatment would not contribute to a different outcome. On the other hand, in the absence of interaction, we have a greater number of samples that endow us a greater degree of freedom that may capture significant differences among genotypes and treatments, as was presented in the current statistical analysis.

Further, this reasoning was adopted and amended in the reviewed version. As mentioned earlier, we have additional data on metabolites that helps understanding possible track of the unexpected similarity between SuzT and SuzS, to date:

• Empirical data base;
• Small number of genotypes (clones) that were approached in this initial experiment;
• Different set of strategies that are enclosed in each genotype coping with water deficit and EPD resistance;
• Unpublished results (ID PHYTOCHEM-D-23-00164) that will be mentioned as such or citing the published article as soon as it is through the reviewing process, that is concomitant with this one.
• Further, there is one reasoning raised in Picoli et al (2021) concerning roots, that is dry matter is not the only adaptation that is acceptable to contribute to water deficit. For instance, we may have the same amount of roots but with different diameter and order (eg. Fitter, 1987) that will contribute to a more efficient root system in absorbing water and nutrients. Unfortunately, it was not evaluated, but is a fair reasoning.

Fitter, A. H. An architectural approach to the comparative ecology of plant root systems. New Phytol. (1987) 106 (Suppl.), 61-77 61

 

When we analyse the results of the susceptible genotype, we observe that it has less leaf dry matter and a smaller diameter. This is to be expected, as it is more susceptible to water deficit and must regulate water loss; however, it is the genotype with the highest photosynthetic rates. How can it show higher photosynthetic rates and, on the other hand, lower biomass? It could be that the control treatment introduces noise into the analysis by increasing the mean photosynthetic values. Therefore, in this case, and as previously mentioned, the results of each genotype in each water deficit situation should be separated. In this way, it will be possible to appreciate the real differences between the genotypes depending on the treatment.  

In our accepted manuscript (PHYTOCHEM-D-23-00164), we observed increased a and b glucose, among other metabolites, accordingly, associated with energy consumption/accumulation or cell wall deposition, respectively. There are common trends in plant metabolism and responses when submitted to water deficit conditions, although they deviate in some point. The higher photosynthetic rates, contrasted with a diminished dry weight, did surprised us as well, but it might be reasoned on the basis of a different destination of the produced photosynthates, photorespiration, for instance.

This reasoning was incorporated in the reviewed manuscript version.

We agree with reviewer 1, the control treatment did imply in noise in the analysis as observed in Corrêa et al (2023). We may either sustain this argument (first option in the reviewed manuscript) or review the analysis to incorporate it as well (second option for a 2^nd review round). Again, if possible, we ask for the extension of the review deadline for conducting and interpreting the analyses again taking this into account.

 

With regard to the second case, grouping the genotypes to observe how they respond to the treatments, we make the same mistake again. This analysis of the data does not provide any meaning. In fact, in almost all the figures comparing treatments, there are no significant differences. This is obviously an error due to the focus of the results.

We argue that, by grouping the genotypes, we have a general trend as an outcome that guides eucalypt clone responses to water deficit, although it is not absolute.

The lack of significant differences is interpreted as a result of a small number of clones sampled. It was amended in the reviewed manuscript.

 

In addition to the above, when you read the keywords, one of them is osmotic stress.

Key word “osmotic stress” was withdrawn.

 

If you look at the material and methods, the heading indicates water potential, and in the results, figure 1 also represents water potential. The authors cannot talk about water potential as they have not measured it. Instead, they have measured the osmotic potential.

The status of water free energy is an interesting theme. As compiled by Taiz and Zeiger (2010) chemical potential is an approach for the free energy associated with a substance, in this case, the water.

Commonly we have three components (Ψs, Ψp, and Ψg) solute, pressure and gravity components of water potential (Ψw). Independently from the system in focus, we may have different water potential components, accordingly, we may have the water potential of a solution (Ψw_s) with a unique component, the solute potential (Ψs), where, in this situation, we will have Ψw_s = Ψs. Still, it can be denominated water potential, as we did.

Considering different systems, such as solutions, soils and plant tissues, we may come across low hydric contents in which we may consider a matric potential (Ψm), in which water is presented in tinny layers, interacting with solid surfaces by electrostatic interactions, and Ψs and Ψp could not be easily differentiated. In our research we approached the amount of water that was available (free energy water) with the cryosmometer and converted it in water potential units (MPa), in other words, the resulting water potential of the water solution that we expected to find in each substrate, for each plant used. To avoid unnecessary explanations on these water potential components, we used only “water potential”. If the reviewer understands it is necessary, we may add this reasoning. This information consists of the end values of water potential eucalypt seedlings found in the substrate for each treatment.

Further, several reports use either water potential (more frequent) or osmotic potential for the identification of this variable.

We amended the explanation for the conversion of the cryosmometer measures into water potential units.

Taiz, L. and Zeiger, E. (2010) Plant Physiology. 5th Edition, Sinauer Associates Inc., Sunderland, 782 p.

 

This also leads to a problem, as there does not have to be a direct relationship between the osmotic potential and the water potential on the plant. Even if there is a lower osmotic potential in the soil, the regulation of the cellular water status by the plant is done through transpiration and compatible osmolytes. This can lead to a substantial change in water uptake, and consequently to significant variations in soil water potential. Therefore, figure 1 and any allusion to water potential should be changed to osmotic potential.

Unfortunately, we have not the water potential estimates for this experiment. Nevertheless, it is presented and discussed in Correa et al (2023) for a wider number of eucalypt clones, and supporting the expectations of reviewer 1.

As explained before and in the material and methods, the measure of water potential that is presented approaches the estimation of the water potential of the solution that is expected to be found in the substrate of each combination of plant and treatment. It was a measure of the water stress provided in each treatment as shown in figure 1.

Certainly, plants will undergo an adaptation to the “osmotic/water potential” environment, although PEG is not expected to be metabolized or absorbed. Several reports (some of them referenced in the following) use PEG, with success, in water deficit stress simulation. If reviewer 1 understands that further explanation is needed, we may provide it in a second round of review.

Unfortunately, we could not manage to have a greater amount of possible combinations of variables in all of our experiments. We hope soon to gather these information and promising characteristics (biomarkers) and test them in a context of a routine of a breeding program and considerably greater number of genotypes.

References:

1. L Nepomuceno, D.M Oosterhuis, J.M Stewart, Physiological responses of cotton leaves and roots to water deficit induced by polyethylene glycol, Environmental and Experimental Botany, Volume 40, Issue 1, 29-41, 1998, https://doi.org/10.1016/S0098-8472(98)00018-5.
2. Hongtao Ji & Xia Li (2014) ABA mediates PEG-mediated premature differentiation of root apical meristem in plants, Plant Signaling & Behavior, 9:11, DOI: 4161/15592324.2014.977720
3. Inès Slama, Tahar Ghnaya, Kamel Hessini, Dorsaf Messedi, Arnould Savouré, Chedly Abdelly, Comparative study of the effects of mannitol and PEG osmotic stress on growth and solute accumulation in Sesuvium portulacastrum, Environmental and Experimental Botany, Volume 61, Issue 1, Pages 10-17, 2007, https://doi.org/10.1016/j.envexpbot.2007.02.004.
4. Khalid A. Khalid, Jaime A. Teixeira da Silva, Weiming Cai, Water deficit and polyethylene glycol 6000 affects morphological and biochemical characters of Pelargonium odoratissimum (L.), Scientia Horticulturae, Volume 125, Issue 2, 159-166, 2010, https://doi.org/10.1016/j.scienta.2010.03.009.
5. Lawlor (1970) observed that only small amounts of PEG were absorbed by intact-root plants and that it can be used for decreasing water potential of plants.
6. Lawlor, D.W. Absorption of polyethylene glycols by plant and their effects on plant growth. New Phytol. 96, 501-513, 1970.
7. Michel, B. E. Evaluation of the water potentials of solutions of polyethylene glycol 8000, both in the absence and presence of other solutes. Plant Physiol, 72, 66-70, 1983.
8. Michel, B. E.; Kaufmann, M. R. The Osmotic Potential of Polyethylene Glycol 6000. Plant Physiol. (1973) 51, 914-916
9. Nelson SK and Oliver MJ (2017) A Soil-Plate Based Pipeline for Assessing Cereal Root Growth in Response to Polyethylene Glycol (PEG)-Induced Water Deficit Stress. Front. Plant Sci. 8:1272. doi: 10.3389/fpls.2017.01272
10. Oqba Basal, András Szabó, Szilvia Veres, Physiology of soybean as affected by PEG-induced drought stress, Current Plant Biology, Volume 22, 100135, 2020, https://doi.org/10.1016/j.cpb.2020.100135.
11. SHEHAB, G. G., AHMED, O. K., & EL-BELTAGI, H. S. (2010). Effects of Various Chemical Agents for Alleviation of Drought Stress in Rice Plants (Oryza sativa L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 38(1), 139–148. https://doi.org/10.15835/nbha3813627
12. Texeira, L. R., Braccini, A. de L. e ., Sperandio, D., Scapim, C. A., Schuster, I., & Viganó, J.. (2008). Avaliação de cultivares de soja quanto à tolerância ao estresse hídrico em substrato contendo polietileno glicol. Acta Scientiarum. Agronomy, 30(Acta Sci., Agron., 2008 30(2)), 217–223. https://doi.org/10.4025/actasciagron.v30i2.1731
13. Verslues PE, Ober ES, Sharp RE. Root growth and oxygen relations at low water potentials. Impact Of oxygen availability in polyethylene glycol solutions. Plant Physiol. 1998 Apr;116(4):1403-12. doi: 10.1104/pp.116.4.1403.
14. Víctor Granda, Candela Cuesta, Rubén Álvarez, Ricardo Ordás, María Luz Centeno, Ana Rodríguez, Juan Pedro Majada, Belén Fernández, Isabel Feito, Rapid responses of C14 clone of Eucalyptus globulus to root drought stress: Time-course of hormonal and physiological signaling, Journal of Plant Physiology, Volume 168, Issue 7, Pages 661-670, 2011, https://doi.org/10.1016/j.jplph.2010.09.015.
15. Villela, F. M. Doni Filho, L., Sequeira, E. L. Tabela de potencial osmótico em função da concentração de polietileno glicol 6000 e da temperatura. Pesquisa Agropecuária Brasileira, 26(11/12): 1957-1968, 1991.

 

In summary, the paper has potential but should be rewritten. The figures in which the treatments are grouped together should be removed, and instead the results of each genotype should be analysed according to the water deficit treatments. Obviously hedgerows means changing the discussion. The approach to osmotic potential should also be rewritten.

The reviewed manuscript has several modifications that we hope satisfy reviewer 1 questions and that aimed redeeming possible doubts. We evaluated the figures and will wait for the considerations of reviewer 1 about our arguments and reasoning. If necessary they will be withdrawn. The same is valid for the osmotic potential x water potential argument.

We will perform the due data reanalysis if reviewer 1 understands that it is still needed.

Reviewer 2 Report

Summary

PEG Solution? What is it?

avoid using the word suggest! Only in the summary it appears 3 times.

Detail how the experiment was carried out, in dic or dbc.

 

Introduction

In which phase does the water deficit affect the most? Important to contextualize.

Currently there are several technologies to reduce this effect and in eucalyptus the use of hydrogels is highlighted. Check this out in the review.

 

Materials and methods

 

The delineation is confusing, only at the end did it become clear.

I strongly suggest describing the design better at the beginning of the section.

Also, isn't it clear what the treatments are? How were the PEGs determined? Describe in detail the PEG!

Also, under what conditions were the experiments carried out? Time of the year? Greenhouse features?

This section should be better described.

 

I suggest a table with the pavlores. right at the beginning of the results, so it will be easier to understand the differences.

Not shown that had no significant difference. There was no interaction of any variable?

I suggest looking for data on PEG in the results. My suggestion is to understand what this is and what its effects are.

 

The discussion section is very well presented.

Author Response

 

PEG Solution? What is it?

It was amended.

 

avoid using the word suggest! Only in the summary it appears 3 times.

It was amended.

 

Detail how the experiment was carried out, in dic or dbc.

It was already mentioned in “Statistical analysis and experimental design:

“The experiment was conducted in a randomized block design in a 4x3 factorial scheme, consisting of four genotypes and three treatments, with five blocks/replicates. Each experimental unit was composed of three plants.

 

 

Introduction

In which phase does the water deficit affect the most? Important to contextualize.

Currently there are several technologies to reduce this effect and in eucalyptus the use of hydrogels is highlighted. Check this out in the review.

We agree with reviewer 2, nevertheless, water deficit has potential decrease in production, dry mass increment during all plant cycle. We inserted a reference on that, but, discussing it further, possible effects on different culture stages, in planted forests or natural forests, use of the hydrogel in the formation/plantation will only increase the length of the article. Particularly hydrogel will have significant effect on seedling being transplanted to field conditions, but not throughout the productive cycle.

Again, if reviewer 2 understands it is imperative, we will insert further references on these topics.

 

Materials and methods

The delineation is confusing, only at the end did it become clear.

The experimental design was relocated to the beginning of the Material and Methods for the sake of a better comprehension of the experimental design separated from the statistical analysis. Further explanation and reorganization will be implemented if reviewer 2 understands it is necessary.

 

I strongly suggest describing the design better at the beginning of the section.

Ok, it was amended.

 

Also, isn't it clear what the treatments are? How were the PEGs determined? Describe in detail the PEG!

We inserted a brief explanation of PEG solution in the manuscript. The use of PEG to simulate water deficit is reported in several articles approaching water deficit simulation. Some of them are mentioned in the replies to reviewer 1 (See in the following). If necessary, there are other articles on this topic available. Some of them were added and discussed in the manuscript to solve reviewer 2 questions. It was amended. 

 

References:

1. L Nepomuceno, D.M Oosterhuis, J.M Stewart, Physiological responses of cotton leaves and roots to water deficit induced by polyethylene glycol, Environmental and Experimental Botany, Volume 40, Issue 1, 29-41, 1998, https://doi.org/10.1016/S0098-8472(98)00018-5.
2. Hongtao Ji & Xia Li (2014) ABA mediates PEG-mediated premature differentiation of root apical meristem in plants, Plant Signaling & Behavior, 9:11, DOI: 4161/15592324.2014.977720
3. Inès Slama, Tahar Ghnaya, Kamel Hessini, Dorsaf Messedi, Arnould Savouré, Chedly Abdelly, Comparative study of the effects of mannitol and PEG osmotic stress on growth and solute accumulation in Sesuvium portulacastrum, Environmental and Experimental Botany, Volume 61, Issue 1, Pages 10-17, 2007, https://doi.org/10.1016/j.envexpbot.2007.02.004.
4. Khalid A. Khalid, Jaime A. Teixeira da Silva, Weiming Cai, Water deficit and polyethylene glycol 6000 affects morphological and biochemical characters of Pelargonium odoratissimum (L.), Scientia Horticulturae, Volume 125, Issue 2, 159-166, 2010, https://doi.org/10.1016/j.scienta.2010.03.009.
5. Lawlor (1970) observed that only small amounts of PEG were absorbed by intact-root plants and that it can be used for decreasing water potential of plants.
6. Lawlor, D.W. Absorption of polyethylene glycols by plant and their effects on plant growth. New Phytol. 96, 501-513, 1970.
7. Michel, B. E. Evaluation of the water potentials of solutions of polyethylene glycol 8000, both in the absence and presence of other solutes. Plant Physiol, 72, 66-70, 1983.
8. Michel, B. E.; Kaufmann, M. R. The Osmotic Potential of Polyethylene Glycol 6000. Plant Physiol. (1973) 51, 914-916
9. Nelson SK and Oliver MJ (2017) A Soil-Plate Based Pipeline for Assessing Cereal Root Growth in Response to Polyethylene Glycol (PEG)-Induced Water Deficit Stress. Front. Plant Sci. 8:1272. doi: 10.3389/fpls.2017.01272
10. Oqba Basal, András Szabó, Szilvia Veres, Physiology of soybean as affected by PEG-induced drought stress, Current Plant Biology, Volume 22, 100135, 2020, https://doi.org/10.1016/j.cpb.2020.100135.
11. SHEHAB, G. G., AHMED, O. K., & EL-BELTAGI, H. S. (2010). Effects of Various Chemical Agents for Alleviation of Drought Stress in Rice Plants (Oryza sativa L.). Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 38(1), 139–148. https://doi.org/10.15835/nbha3813627
12. Texeira, L. R., Braccini, A. de L. e ., Sperandio, D., Scapim, C. A., Schuster, I., & Viganó, J.. (2008). Avaliação de cultivares de soja quanto à tolerância ao estresse hídrico em substrato contendo polietileno glicol. Acta Scientiarum. Agronomy, 30(Acta Sci., Agron., 2008 30(2)), 217–223. https://doi.org/10.4025/actasciagron.v30i2.1731
13. Verslues PE, Ober ES, Sharp RE. Root growth and oxygen relations at low water potentials. Impact Of oxygen availability in polyethylene glycol solutions. Plant Physiol. 1998 Apr;116(4):1403-12. doi: 10.1104/pp.116.4.1403.
14. Víctor Granda, Candela Cuesta, Rubén Álvarez, Ricardo Ordás, María Luz Centeno, Ana Rodríguez, Juan Pedro Majada, Belén Fernández, Isabel Feito, Rapid responses of C14 clone of Eucalyptus globulus to root drought stress: Time-course of hormonal and physiological signaling, Journal of Plant Physiology, Volume 168, Issue 7, Pages 661-670, 2011, https://doi.org/10.1016/j.jplph.2010.09.015.
15. Villela, F. M. Doni Filho, L., Sequeira, E. L. Tabela de potencial osmótico em função da concentração de polietileno glicol 6000 e da temperatura. Pesquisa Agropecuária Brasileira, 26(11/12): 1957-1968, 1991.

 

Also, under what conditions were the experiments carried out? Time of the year? Greenhouse features?

This section should be better described.

It was amended.

 

I suggest a table with the pavlores. right at the beginning of the results, so it will be easier to understand the differences.

It was amended.

 

Not shown that had no significant difference. There was no interaction of any variable?

Supplemental table S1 shows the ANOVA for the genotypes and treatments. There were significant treatment and genotype effects, although only Sulphur, copper and amino acid content displayed significant interaction between genotype and treatment.

 

I suggest looking for data on PEG in the results. My suggestion is to understand what this is and what its effects are.

It was amended.

 

The discussion section is very well presented.

We thank reviewer 1 and 2 for the comments. We hope we have fulfilled their comments or redeemed their considerations that needed to be improved. If necessary, further amendments will be provided in a second review round.

Round 2

Reviewer 1 Report

The paper presented by Madeira et al. has been modified to a great extent, trying to solve the shortcomings previously mentioned in the first review. In my first review I already commented on two important points, the water potential and the grouping of the data by genotypes and treatments. The rest of the comments have been satisfactorily corrected. I will now comment on the two most important aspects.

 

With regard to the grouping of treatments, the authors comment that there is no interaction between the variables, so the treatments can be grouped together. In those cases where there was interaction, they were separated. Indeed, the authors are right, and in that case it can be done. The justification for doing it this way, and I cite, "Since we had no interaction and, a considerable number of the traits showed no significant differences, breaking down the results by genotype and treatment would not contribute to a different result. On the other hand, in the absence of interaction, we have a larger number of samples that provide us with a greater degree of freedom that can capture significant differences between genotypes and treatments, as presented in the current statistical analysis".

 

In my opinion, statistics is a tool, and should be used as such, not as an end in itself. The authors should take a larger number of samples and design the experiment better in order not to encounter this problem. Although statistically this approach can be done, the question is whether it really represents the reality and the aim we are trying to achieve. In my opinion, this type of clustering, although correct, is an approach that is not optimal for knowing what is actually happening.

Regarding the second point, water potential, I do not agree with the authors' estimation. Firstly, the reference they use to explain the change from osmotic potential to water potential is an academic textbook, but not scientific per se (Taiz and Zeiger, 2010). Secondly, I mention an article to transform °H into MPa. The article in question (Szijarto & van de Voort, 1983) deals with cow's milk and goat's milk, and at no point in the text is MPa mentioned, but rather the conversion of °C to °H.

 

On this point I cannot agree with the authors, as the solute content cannot be equated to the water potential of the plant. First of all, which plant substrate is chosen? Is it leaves, roots... It is not developed in the material and methods. Secondly, if it is plant material, why do the authors estimate that the pressure potential is zero. Plant cells have turgor potential, which undoubtedly influences the water potential, and this difference can be very large depending on the tissue, organ and cell type of the plant. With this procedure, it is not possible to know or estimate the water potential in any way. Undoubtedly the PEG treatment must dry out the soil and generate a water deficit, but we can neither calculate nor estimate the water potential. It would be better to try to calculate the water deficit of the soil by the use of PEG than what is presented in the paper, and present the results on that basis, something the authors could do in a short period of time.

Ultimately, I think that the water potential section should be changed and try to express it in a different way. Regarding the first point, this is a matter for the editor to assess.

Author Response

Dear Reviewer 1,

 

We are grateful and indebted with you for your comments that helped us improve our manuscript.

 

We updated the manuscript and the  response to your comments in this 2^nd review round, and further amendments were performed in this updated version. 

 

The paper presented by Madeira et al. has been modified to a great extent, trying to solve the shortcomings previously mentioned in the first review. In my first review I already commented on two important points, the water potential and the grouping of the data by genotypes and treatments. The rest of the comments have been satisfactorily corrected. I will now comment on the two most important aspects.

We thank reviewer 1 for his/her comments. We further approach the important topics mentioned above.

 

With regard to the grouping of treatments, the authors comment that there is no interaction between the variables, so the treatments can be grouped together. In those cases where there was interaction, they were separated. Indeed, the authors are right, and in that case it can be done. The justification for doing it this way, and I cite, "Since we had no interaction and, a considerable number of the traits showed no significant differences, breaking down the results by genotype and treatment would not contribute to a different result. On the other hand, in the absence of interaction, we have a larger number of samples that provide us with a greater degree of freedom that can capture significant differences between genotypes and treatments, as presented in the current statistical analysis".

In my opinion, statistics is a tool, and should be used as such, not as an end in itself. The authors should take a larger number of samples and design the experiment better in order not to encounter this problem. Although statistically this approach can be done, the question is whether it really represents the reality and the aim we are trying to achieve. In my opinion, this type of clustering, although correct, is an approach that is not optimal for knowing what is actually happening.

We agree that the use of a greater number of samples sure will help future experiments. Considering our possibilities, we will have this in mind. Unfortunately, we do not have further samples or repetitions available for this experiment. This was the second experiment we conducted in this project in which one of our goals was to northern the choice of biomarkers associated with the tolerance trait and, if possible, further contribute to the understanding of the tolerant phenotype. Being that said, we acknowledge that there are issues that were missing, due it is an initial approach for the metabolic profile. We tried to fulfill some of the issues highlighted by reviewer 1 in the ulterior experiments, such as the assessment of leaf water potentials as in Corrêa et al 2023. Additionally, we mentioned another article that is under review, which results we quote as “data not published”. This mentioned article results were derived from an experiment performed after the one we show in the present report, but helped us with information on the metabolic behavior of contrasting genotypes conducted under water deficit stress. Up until now, we are waiting for the final acceptance of this other article, reason why this information was not updated in this last version of our manuscript in review at Agronomy.

 

Regarding the second point, water potential, I do not agree with the authors' estimation. Firstly, the reference they use to explain the change from osmotic potential to water potential is an academic textbook, but not scientific per se (Taiz and Zeiger, 2010).

We understand and respect reviewer 1 argument and followed his/her recommendation for the use of osmotic potential, despite we also have arguments from scientific articles adopting and using the alternative form for denomination of water potential. The recommended terminology was applied in this reviewed version of the manuscript.

 

Secondly, I mention an article to transform °H into MPa. The article in question (Szijarto & van de Voort, 1983) deals with cow's milk and goat's milk, and at no point in the text is MPa mentioned, but rather the conversion of °C to °H.

On this point I cannot agree with the authors, as the solute content cannot be equated to the water potential of the plant. First of all, which plant substrate is chosen? Is it leaves, roots... It is not developed in the material and methods. Secondly, if it is plant material, why do the authors estimate that the pressure potential is zero. Plant cells have turgor potential, which undoubtedly influences the water potential, and this difference can be very large depending on the tissue, organ and cell type of the plant. With this procedure, it is not possible to know or estimate the water potential in any way. Undoubtedly the PEG treatment must dry out the soil and generate a water deficit, but we can neither calculate nor estimate the water potential. It would be better to try to calculate the water deficit of the soil by the use of PEG than what is presented in the paper, and present the results on that basis, something the authors could do in a short period of time.

We do not refer to the plant water potential. From the very beginning manuscript, although unusual, we mentioned estimating the osmotic potential (term adopted in this reviewed version) of the solution in the substrate in which the plants were grown during the experiment, although we expect that the differences observed did influence the water potential in the plants.

Please, observe Materials and Methods (first submitted version): “The water potential of the substrate solution of each pot where the plants were grown was estimated at the end of the experiment to verify the water deficit applied to the plants.”, and in the last (revised version): “The osmotic potential of solution of the substrate of each pot where the plants were grown was estimated at the end of the experiment to verify the water deficit applied to the plants.”

The potential near zero was observed for the control treatment. It was observed for the solution obtained individually from the recipients (2 L plastic bags), in a standardized form described in the manuscript, from the commercial substrate (updated in the reviewed manuscript) where the plants were conducted during the experiment, and for all treatments. The other treatments achieved potentials around -0.3 and -0.5 MPa, for 100PEG and 300PEG, respectively. These results are depicted as “estimated osmotic potential of the substrate solution of substrate in the pots where the plants were conducted in the experiment with three different treatments of osmotic stress.” in this reviewed version.

We recognize that a full conversion steps and explanation was missing in the former manuscript version. It was amended. Considering it may turn the manuscript wordy, the references, although cited in the manuscript, were informed in the text, and the steps explained in detail in the Supplemental Methods 1. Despite unusual making use of procedures from articles dealing with food products and plant propagation samples, the conversion steps to transform °H into MPa was useful in our research and these detailed, and referenced steps, may help other researchers in their experiments and analysis if similar approaches are demanded.

This is reasoned as there are estimates of PEG solution osmotic potentials, as a criterion for approaching water deficit (Please, see Michel and Kaufmann (1973) and Villela et al (1991)), but we used PEG solution treatment (described in the reviewed manuscript Material and Methods) concomitant with the fertilization and watering the plants schedule, during the experiment. This procedure, as expected and observed in figure 1, achieved a lower osmotic potential in the substrate (where the plants were grown) different from the estimate for the isolated PEG solution estimate. Independently from this reasoning, the treatment was successful in reducing the osmotic potential, and by consequence, simulating water deficit stress.

As mentioned in the previous review round, unfortunately, we do not have the data on the plant water potential estimates for this experiment. Nevertheless, it is presented and discussed in other experiment report (Correa et al 2023) for a wider number of eucalypt clones, and supporting the expectations of reviewer 1.

We are sorry if we lead to a misunderstood of our approach. Again, unfortunately, we do not have the water potential approach for “plant samples” in this experiment. It was provided in other experiment (Corrêa et al 2023). We amended the terminology in this revised version. Our approach was exactly to have an estimate the water deficit in the standardized solutions extracted from the substrate where the plants were grown during the experiment and designated it, as suggested by reviewer 1, as osmotic potential of the substrate in this reviewed version.

This was an attempt to approach the water stress that was conditioned in the substrate plants were grown. This water stress simulation provided by the use of PEG solution is acknowledged in several scientific articles as already mentioned before. We inserted four reports (articles), but there are others that may be cited and that use PEG solutions for simulating and achieving water deficit conditions/treatments.

Please, observe that Michel and Kaufmann (1973) and Villela et al (1991) already estimated the osmotic potential of PEG 6000 solutions. Indeed, in these articles, we have the reference of osmotic potential of these solutions and the associated water stress.

The data set that was analyzed and shown in the present report is the data set we obtained. We have learned from this experience and foresaw the water potential estimation from leaf samples for ulterior experiments, as exemplified in Corrêa et al (2023).

Despite our arguments, we understand that we use information that is compiled in a referenced text book (George et al 2008) and that there are several steps (described in this reviewed version) to achieve the MPa estimates that, for the sake of comprehension might difficult this approach. This was amended and the procedures and references presented in detailed steps that may guide anyone that might be interested in repeating this process. If reviewer 1 and the editor understand this is an unsuitable approach, we may withdraw the information on the water deficit measure approximation in a 3^rd review round.

Despite our justification, we must mention that we used the information from George et al (2008), to perform step 4 (please see reviewed supplementary material) of the conversion from oH to MPa. We are aware that there are scientific articles that used the approach to perform this conversion in another situation (Ribeiro et al 2009; Dorneles et al 2021), but we tried to be as straight as possible describing our procedures allowing anyone to follow it in a ulterior experiment. We reiterate, we seek and presented a series of procedures, used in other experiments although with different specimens and origins, to extract the most of the information we understood was possible. We strived for these goals based on the information and data we had from the experiment we are presenting in this report. We are open to the critics and suggestions that warned us on our flaws and thank reviewer 1 for helping us improving our manuscript.

 

Villela, F. M. Doni Filho, L., Sequeira, E. L. Tabela de potencial osmótico em função da concentração de polietileno glicol 6000 e da temperatura. Pesquisa Agropecuária Brasileira, 26(11/12): 1957-1968, 1991.

Michel, B. E.; Kaufmann, M. R. The Osmotic Potential of Polyethylene Glycol 6000. Plant Physiol. (1973) 51, 914-916

Baer, R.J; Baldwin, K.A. Freezing Points of Bulking Agents Used in Manufacture of Low-Calorie Frozen Desserts. J. Dairy Sci 1984, 67(12): 2860-2862. https://doi.org/10.3168/jds.S0022-0302(84)81647-1.

Henriques, G.S.; Rosado, G.P. Formulação de dietas enterais artesanais e determinação da osmolalidade pelo método crioscópico. Rev Nutri 1999, 12(3): 225–232. https://doi.org/10.1590/S1415-52731999000300003

George, E.F.; Hall, M.A.; De Klerk, G.J. Plant growth regulators I: Introduction; auxins, their analogues and inhibitors. In: Plant Propagation by Tissue Culture; Springer: Dordrecht, The Netherlands, 2008; pp. 115–174.

Dorneles, A.O.S., Pereira, A.S., da Silva, T.B. et al. Responses of Solanum tuberosum L. to Water Deficit by Matric or Osmotic Induction. Potato Res. 64, 515–534 (2021). https://doi.org/10.1007/s11540-020-09489-3

Ribeiro, J. P. N., Matsumoto, R. S., Takao, L. K., Voltarelli, V. M., & Lima, M. I. S.. (2009). Efeitos alelopáticos de extratos aquosos de Crinum americanum L.. Brazilian Journal of Botany, 32(1), 183–188. https://doi.org/10.1590/S0100-84042009000100018

 

Ultimately, I think that the water potential section should be changed and try to express it in a different way. Regarding the first point, this is a matter for the editor to assess.

As we mentioned in the previous review round, we argued one point of view, but, we understand the perspective of reviewer 1, and present a revised approach expressing the possible considerations on water potential. We hope we have contemplated comments and redeemed reviewer 1 questions in this reviewed version. 

 

With regards,

 

 

Reviewer 2 Report

Dear Editor, 

The author realizes all corrections. 

Author Response

Dear reviewer 2,

 

We are grateful and indebted with you for your comments that helped us improve our manuscript. 

In response to reviewer 1 comments in a 2 review round, further amendments were performed in the updated version. 

 

With regards,