Effects of Summer Water Deficit Stress on Olive Fruits and Oil Quality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The reviewed article horticulturae-2864015 entitled “Effects of summer water deficit stress on olive fruits and oil quality” raises an interesting and timely topic of the impact of climate change on the quality of crops. The cultivation of olive trees and oil production is an economically significant product of the Mediterranean climate zone, so research in this area is of great importance. In my opinion, the work was well prepared and presented. I did not find any errors requiring correction. However, I would have two suggestions. I would suggest that when presenting results regarding the analysis of bioactive compounds, use the term "concentration" and not "content". This term, in my opinion, better reflects the results of these analyses. The second issue is the presentation of the results of the concentration of bioactive compounds. In my opinion, it would be better to present the results in terms of oil volume or dry/fresh weight of olives rather than relative peak areas. The results presented in this way illustrate the differences between the tested combinations, but it is more difficult to compare them with the published results of other researchers because this form is rarely used. These comments, however, do not affect my high rating of the manuscript and are of a more advisory nature.  Since my opinion about the reviewed work is positive, I believe that it can be published in its current form.

Author Response

Reviewer #1:

Comment: The reviewed article horticulturae-2864015 entitled “Effects of summer water deficit stress on olive fruits and oil quality” raises an interesting and timely topic of the impact of climate change on the quality of crops. The cultivation of olive trees and oil production is an economically significant product of the Mediterranean climate zone, so research in this area is of great importance. In my opinion, the work was well prepared and presented. I did not find any errors requiring correction. However, I would have two suggestions. I would suggest that when presenting results regarding the analysis of bioactive compounds, use the term "concentration" and not "content". This term, in my opinion, better reflects the results of these analyses. The second issue is the presentation of the results of the concentration of bioactive compounds. In my opinion, it would be better to present the results in terms of oil volume or dry/fresh weight of olives rather than relative peak areas. The results presented in this way illustrate the differences between the tested combinations, but it is more difficult to compare them with the published results of other researchers because this form is rarely used. These comments, however, do not affect my high rating of the manuscript and are of a more advisory nature.  Since my opinion about the reviewed work is positive, I believe that it can be published in its current form.

Response: We acknowledge the positive comments about our work. We understand the opinion of the Reviewer to replace of the term “content” by “concentration”, but taking in to about that semi-quantification procedure calculate the relative abundance, we replace “content or level” by “abundance”.

Reviewer 2 Report

Comments and Suggestions for Authors

This work aims to study the effect of the stressful conditions of a dry year (2017) on the quality of the fruit and olive oil. The topic of work is not new since there is a considerable number of articles in the bibliography that address the effect of water deficit on different quality parameters of olives and oil. To do this, it is carried out with strict control of the irrigation water supply or measurement of the water level in the soil. This is something that is missing in this work and, consequently, is one of the main concerns presented in the article. In Materials and Methods it is said that 'The selected orchard was monitored from June to the end of September' (lines 90-91), however no monitoring data is provided such as temperature, rainfall, soil humidity, evapotranspiration, water potential parameters of the plant, etc. There is no objective data, only subjective assessments of the state of the plant. Nor is the level of ripening of the fruits indicated, nor is it clear the number of trees in the orchard under study, or the number of oil extractions carried out. From what is described in lines 110-111, apparently only five trees per area have been studied, extracting a single oil per tree. This could not be considered representative for a study of the effect of the weather in 2017 on the quality of the olive and its oil.

On the other hand, in relation to the description of the analyses carried out, the protocols and techniques used are described, but they should be clearly indicated for which metabolites they were intended for.

Finally in the Materials and Methods section, it is described that the olive fruits were destoned and milled (line 165), after which they were extracted with hexane for analysis of fatty acids and the residue with methanol for extraction of the phenolic fraction. According to my experience, while there would be no problem in the first case, the grinding of the fruit prior to extraction entails a complete alteration of the phenolic profile due to hydrolysis (mainly due to beta-glucosidase activities) and oxidation reactions (especially due to polyphenol oxidases). See for example the low proportion that oleuropein presents in Table 1, when it is fully established that it is by far the main compound in olives.

 Regarding the results and discussion thereof, there is a problem of interpretation. Changes in the content of some phenolic compounds are reported (see tables 1 and 2) based on the percentages of the total area in their analyses, that is, they are increases and decreases in the proportion of these compounds within each sample. Consequently, they do not necessarily mean differences in the content of these compounds between hydrated and dry soil area samples. It is necessary to present quantitative data for each of these compounds to be able to make comparisons between samples from the dry and hydrated areas of the orchard. For this, the external standard could be used for a quantitative estimate, which theoretically was used as indicated in Materials and Methods.

 Other issues that authors should consider are the following:

Line 203 - As described in Materials and Methods, the chromatograms obtained were analyzed at 280, 240 and 230 nm, but the semi-quantification of the individual compounds was performed by peak integration at 240 nm. Although it would be correct for quantification of compounds with a secoiridoid structure, it would not be appropriate for phenolic derivatives (280 nm is best) or flavonoids (340 nm is best). Why are the different detections not used for each group of compounds?

Line 370 - The main vitamer in olives and olive oil is alpha-tocopherol, not the beta form.

Figure 3 – In my opinion there is a problem in the identification of alpha-tocopherol in oils. Alpha-tocopherol should appear, especially if there is so much in the fruit.

Figure 4 - Omit. As it stands, it looks like the origin of the fragments in the MS spectrum of oleuropein.

Author Response

Comment 1: This work aims to study the effect of the stressful conditions of a dry year (2017) on the quality of the fruit and olive oil. The topic of work is not new since there is a considerable number of articles in the bibliography that address the effect of water deficit on different quality parameters of olives and oil. To do this, it is carried out with strict control of the irrigation water supply or measurement of the water level in the soil. This is something that is missing in this work and, consequently, is one of the main concerns presented in the article. In Materials and Methods it is said that 'The selected orchard was monitored from June to the end of September' (lines 90-91), however no monitoring data is provided such as temperature, rainfall, soil humidity, evapotranspiration, water potential parameters of the plant, etc. There is no objective data, only subjective assessments of the state of the plant. Nor is the level of ripening of the fruits indicated, nor is it clear the number of trees in the orchard under study, or the number of oil extractions carried out. From what is described in lines 110-111, apparently only five trees per area have been studied, extracting a single oil per tree. This could not be considered representative for a study of the effect of the weather in 2017 on the quality of the olive and its oil.

Response: The authors thank the Reviewer comment. The aim is to work with field trees exposed to a real scenario of extreme weather conditions (heat and drought). We agree that there is missing information, and we added a new figure (Figure 1, in the new version of the manuscript) with information concerning the climatic conditions collected at a meteorological station located next to the selected plots (please see lines 96-99, pg. 2). Parameters such as temperature (minimum - TMIN, average - TAVE and maximum - TMAX), and precipitation were evaluated in the harvest year (2017). Moreover, tree physiology and oxidative stress responses (like plant water status, antioxidant enzymes, membrane damages, ROS, and phenolic and lipophilic leaf profiles) were also evaluated and data was published previously at Araújo et al. 2021 (reference number 18). Regarding the number of trees studied and the ripening stage of the fruits, five blocks of ten plants were selected in both plots, hydrated and dry, and fruits were harvested per plant at a state of maturation that varied between M2.5 and M3 in accordance with the guidelines defined by the International Olive Oil Council (https://www.internationaloliveoil.org/wp-content/uploads/2019/11/COI-OH-Doc.-1-2011-Eng.pdf). We added this information to the manuscript, please see lines 102-108, pg 3.

 

Comment 2: On the other hand, in relation to the description of the analyses carried out, the protocols and techniques used are described, but they should be clearly indicated for which metabolites they were intended for.

Response: We understand the Reviewer comment. We added the information about the compounds identified for each chromatography method. Please see pgs. 4 to 5.

 

Comment 3: Finally in the Materials and Methods section, it is described that the olive fruits were destoned and milled (line 165), after which they were extracted with hexane for analysis of fatty acids and the residue with methanol for extraction of the phenolic fraction. According to my experience, while there would be no problem in the first case, the grinding of the fruit prior to extraction entails a complete alteration of the phenolic profile due to hydrolysis (mainly due to beta-glucosidase activities) and oxidation reactions (especially due to polyphenol oxidases). See for example the low proportion that oleuropein presents in Table 1, when it is fully established that it is by far the main compound in olives.

Response: We appreciate the reviewer's comment. We agree with the reviewer's point of view, however the time that elapsed between removing the seed, crushing the sample and mixing it with the eluent was relatively short. We believe that extraction was not the main factor that led to the lowering of oleuropein. Furthermore, milling is an important process to free up the compounds to be extracted (fatty acids and phenols). These low levels of some compounds could be due to the early harvest, which probably meant that all the compounds had not yet been formed in the appropriate quantities.

 

Comment 4: Regarding the results and discussion thereofe, there is a problem of interpretation. Changes in the content of some phenolic compounds are reported (see tables 1 and 2) based on the percentages of the total area in their analyses, that is, they are increases and decreases in the proportion of these compounds within each sample. Consequently, they do not necessarily mean differences in the content of these compounds between hydrated and dry soil area samples. It is necessary to present quantitative data for each of these compounds to be able to make comparisons between samples from the dry and hydrated areas of the orchard. For this, the external standard could be used for a quantitative estimate, which theoretically was used as indicated in Materials and Methods.

Response: We started with preparing the extract (section 2.4.1.) using similar material for both conditions (hydrated and dry). Then, the quantity of extract used for each condition (hydrated and dry) and injected (1 µL) in the chromatography equipment, GC-MS or UHPLC-MS, was similar. This is referred to in M&M (lines 164 to 204). The extracts of both conditions prepared for GC-MS were 40 mg mL-1 for fruits and 20 mg mL-1 for oil, and the extract for the UHPLC-MS was also 40 mg mL-1 for fruits and 30 mg mL-1 for oil. The analysis of the chromatograms obtained was performed by choosing the same number of peaks (that correspond to the metabolites identified) in both conditions. The compound relative abundance was then determined [relative peak area average (%) = (compound peak area/total peak area average) * 100]. This allows a comparison between conditions since we started with the same concentrations and injected the same volume for analysis. The use of the relative abundance percentage method was already performed before in our previous work (https://dx.doi.org/10.1021/acs.jafc.0c04719) or by other colleagues (e.g., https://doi.org/10.1016/j.ecolind.2022.109315; https://doi.org/10.1186/s12870-019-2231-y). In the new version of the manuscript, we explained better how the semi-quantitative analysis was performed (pg. 5, lines 222-233). We replaced the terms “level of metabolites” and “content metabolites” by “abundance”, referring to the relative abundance percentage of each compound.

 

Comment 5: Other issues that authors should consider are the following:

Line 203 - As described in Materials and Methods, the chromatograms obtained were analyzed at 280, 240 and 230 nm, but the semi-quantification of the individual compounds was performed by peak integration at 240 nm. Although it would be correct for quantification of compounds with a secoiridoid structure, it would not be appropriate for phenolic derivatives (280 nm is best) or flavonoids (340 nm is best). Why are the different detections not used for each group of compounds?

Response: We apologise for the misunderstanding. The individual compounds' semi-quantification in the fruit and oil extracts was performed by peak integration at the appropriate wavelength. We correct the manuscript accordingly (please see line 227-228 “The semi-quantification of the individual compounds in the fruit and oil extracts was performed by peak integration at the appropriate wavelength.”).

 

Comment 6: Line 370 - The main vitamer in olives and olive oil is alpha-tocopherol, not the beta form.

Response:  Thank you for the correction, we correct this point in the Discussion section (please see line 403, pg 11).

 

Commet 7: Figure 3 – In my opinion there is a problem in the identification of alpha-tocopherol in oils. Alpha-tocopherol should appear, especially if there is so much in the fruit.

Response: Usually, alpha-tocopherol appears in olive oils, but in fact, the abundance in our fruits in hydrated conditions is very low, ~0.3%, which, putatively, is even lower in oils and undetectable in our samples. The decomposition of this compound in other metabolites can also occur and justify the low or undetectable levels.

 

Comment 8: Figure 4 - Omit. As it stands, it looks like the origin of the fragments in the MS spectrum of oleuropein.

Response: We followed the suggestion of the Review, and we excluded the figure 4.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is interesting and showed good goals to understand the quality and chemical composition of different olive oils and it’s influence by the climate conditions .

Line 43: “fruits” are referred to olives, please clear this sentence

Line 70-74: this is a hypothesis and can be linked to the concepts and literature about this them in Introduction section. As suggestion, in Introduction section, the authors needed detailed about the climate conditions and the influence in olive fruits and oils quality

Line 90-91: is necessary a specification about the year of olive fruits cultivation, are not clear, the study was realized in 2017, please this description are needed. Also the authors can indicate the time/year of olive fruits harvest and the maturation index

Line 450-451: in Conclusion the authors affirm that the extreme weather events are responsible for olive trees stress, but in the study does not demonstrate directly this effect, please review this sentence.

Author Response

Comment 1: The manuscript is interesting and showed good goals to understand the quality and chemical composition of different olive oils and it’s influence by the climate conditions .

Response: We acknowledge the positive comments about our work.

 

Comment 2: Line 43: “fruits” are referred to olives, please clear this sentence

Response: Yes, fruits are the olives. We corrected the sentence replacing the name “fruits” by “olives”. Please see lines 44 and 46.

 

Comment 3: Line 70-74: this is a hypothesis and can be linked to the concepts and literature about this them in Introduction section. As suggestion, in Introduction section, the authors needed detailed about the climate conditions and the influence in olive fruits and oils quality.

Response: We added more information about the influence of climate conditions/abiotic stresses effects on olive fruits and oils quality. Please see lines 64 to 74, pg 2.

 

Comment 4: Line 90-91: is necessary a specification about the year of olive fruits cultivation, are not clear, the study was realized in 2017, please this description are needed. Also the authors can indicate the time/year of olive fruits harvest and the maturation index.

Response: We acknowledge the suggestions made by the Reviewer and we added this information.  Regarding the time/year of olive fruits harvest (time between 14-16h, second day of October 2017) this description was added to the manuscript, please see lines 102 and 109. The maturation index varied between M2.5 and 3 (please see line 105, pg. 3).

 

Comment 5: Line 450-451: in Conclusion the authors affirm that the extreme weather events are responsible for olive trees stress, but in the study does not demonstrate directly this effect, please review this sentence.

Response: We agree with the comment of the Reviewer, since in this work we do not focus on the effects of stress on olive stress. We reviewed this sentence (please see lines 479 and 480, pg 12).

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Enclosed

Comments for author File: Comments.pdf

Author Response

Comment 1: For the future, I would recommend the authors to extract phenolics from frozen fruits with liq N2 rather than from fresh fruits, as they used in their work with olive leaves (reference 18). Once fresh fruit is ground, it takes only a few seconds for the phenolics to be hydrolyzed and partially oxidized. This is what happens during the industrial milling of olives in the oil extraction process and the reason for the different phenolic profile in the olive fruit and oil. Finally, regarding the phenolic content in the fruit, they are high during fruit growth and then decrease rapidly before and during ripening (see for example Amiot et al, doi:10.1021/jf00071a014; Bodoira et al, doi:10.1007/s11746-016-2877-7; or Guodong et al, doi:10.1016/j.foodchem.2019.125246).

Comment 2: I would have preferred to see quantitative data to better compare between agronomic conditions as the authors did with leaves (Ref. 18)

Response to comments 1 and 2: We acknowledge the Reviewer comment and recommendation for future work.

 

Comment 3: Actually, the tocopherol content in fruits is ~0.01% according to my data, but this amount yields 100-1000 μg of tocopherols/g of oil after oil extraction. Again, in my opinion, there is a metabolite identification problem. Not only because of tocopherol, but because of others. For example, the high level of cis-vaccenic acid among the fatty acids of the triglycerides in the oil is striking. The fatty acid profile of the oil should almost mimic that of the fruit. An explanation should be included on this point. Furthermore, it is also striking that the proportion of palmitic and oleic acids is quite similar among the triglycerides of the fruit.  

Response: We understand the reviewer comment. Taking in to account these comments we decided to remove the data related to this analysis (fatty acids, tocopherol and squalene) to prevent from putative misunderstood. We removed this data and rearranged the text. Please see the new version of the manuscript.

 

Other points that authors should take into consideration:

Line 103.- Include ‘… based on tree water status determination [18]’

Line 186.- I would change to ‘for the acidic composition by gas chromatography-mass spectrometry (GC-MS) analysis’

Line 192.- I would change to “Olive oil: For the acidic composition by GC-MS analysis, 20 mg …”

Line 193.- I would change to ‘For phenolic analysis by UHPLC-MS, …’

Line 193.- It should be 5 g of olive oil, right?

Line 203.- I guess it is 50 μL of dichoromethane

Line 209.- Change ‘(2021)’ to ‘[18]’

Lines 209-212.- Rewrite and delete ‘lipophilic’, sugars certainly are not

Line 370.- Change ‘levels’ to ‘proportions’

Lines 396-398.- Rewrite the sentence

Response: We corrected the points highlighted above by the Reviewer. We highlighted in the new version of the manuscript, in yellow colour, the parts changed according to the Reviewer comment.