Metabolite Diversity in Pulp Segments, Peel, Leaves, and Bark of a Red-Fleshed ‘Baya Marisa’ Apple Cultivar
, Aljaz Medic 2, Aleks Bordon 3, Metka Hudina 2, Robert Veberic 2, Jerneja Jakopic 2 and Franci Stampar 2
Round 1
Reviewer 1 Report
This study investigated "Metabolite diversity in pulp segments, skin, leaves and bark of a red-fleshed 'Baya Marisa' apple cultivar". The findings are average, no interesting innovations. However, the following comments should be introduced in the manuscript:
- The abstract is not well organized and not resumes the findings of the research.
- The introduction is well structured, the author presented the recent findings, established the research gap, and clearly stated the research objectives. But why authors have chosen the apple cultivar as their material? Write few lines for it.
- Provide a strong hypothesis at the end of introduction section.
- Page 1 line 37 "Malus domestica" shoul be in italic
- Page 3 line 127 please correct "H2SO4"
- Page 3 section 2.3 Please write in LOD: limit of detection; LOQ: limit of quantitation values for individual sugars and organic acids.
- Page 4 section 2.4 Phenolic compounds are oxidized very quickly during and after extraction, especially in apple cultivars due to the high-activity PFO enzyme. Therefore, samples should be boiled in methanol at the extraction stage and antioxidants such as BHT should be added to the samples.
- Page 5 Sorbitol and mannitol, fructose and glucose are compounds that are difficult to distinguish from each other in chromatographic analysis. Therefore, the author should provide the sample or standard chromatogram.
- The conclusion should not be a mere repetition of the results. Instead, consider making it more concise and focused on demonstrating the practical implications of your findings.
-
Although the manuscript is well written, it is verbose.
Author Response
Dear reviewer,
Thank you for your useful comments and suggestions for improvements of our work. We have answered your queries and corrected the text accordingly (the added text or altered sentences are marked with tracking tool). The answers to your comments are listed in a separate file.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript authors investigated the composition of different tissues (pulp, skin, leaves and bark) of 'Baya Marisa', which is a red-fleshed apple cultivar. Although the study was primary aimed to the characterization of this cultivar, imposing a relatively low scientific value of the manuscript, the paper is very well and concisely written, and the composition of both primary and secondary metabolites is presented in detail, and thoughtfully discussed. Based on this, I suggest to publish it in Agriculture journal after addressing the following comments:
Lines 37, 233 and 299: Write the latin names of the species in italic.
Line 71: Change the term ‘display’ with some other more adequate term.
Line 171: Change the term ‘commander’ with some other more adequate term.
Table 1: If you use the abbreviations in the table, the full name has to be given below the table. This applies to P1, P2 and P3, but also to FW (as the table has to be self-explanative).
Table 2: Increase the font of the words, and give the full names for all the abbreviations. Also, for the last four columns, put flesh and skin next to each other as they are part of the same organ.
Table 3: Give the full names for all Pulp 1, Pulp 2 and Pulp 3.
Line 394: Improve the text for ‘that can benefit our health’.
Author Response
Dear reviewer,
Thank you for your useful comments and suggestions for improvements of our work. We have answered your queries and corrected the text accordingly (the added text or altered sentences are marked with tracking tool). The answers to your comments are listed below:
Lines 37, 233 and 299: Write the latin names of the species in italic.
Thank you for pointing out our mistakes. The text has been corrected.
Line 71: Change the term ‘display’ with some other more adequate term.
The term display was changed to present.
Line 171: Change the term ‘commander’ with some other more adequate term.
R commander is the name of the software and cannot be changed with another term (https://en.wikipedia.org/wiki/R_Commander).
Table 1: If you use the abbreviations in the table, the full name has to be given below the table. This applies to P1, P2 and P3, but also to FW (as the table has to be self-explanative).
Abbreviation P1, P2 and P3 were explained in the footnotes of all tables and Figures, where applicable. However, the abbreviation FW (fresh weight) is so standardized that it has been mentioned in the methods (L139) and therefore it seems redundant to include it in footnotes of the tables as well. If the editor feels that it needs explanation, we can of course add it.
Table 2: Increase the font of the words and give the full names for all the abbreviations. Also, for the last four columns, put flesh and skin next to each other as they are part of the same organ.
The name of Table 2 was altered to Tentative identification of 35 phenolic compounds in different parts of 'Baya Marisa', and the standard compounds used for their quantification. We have accidentally copied the name of another table and noticed our mistake. Abbreviations used in column names are standard nomenclature in MS identification and therefore, we feel that they do not need additional explanation. If the editor feels that it needs further definition, we can of course do it.
Table 3: Give the full names for all Pulp 1, Pulp 2 and Pulp 3.
Corrected.
Line 394: Improve the text for ‘that can benefit our health’.
The text was altered to: can improve our health status.
Reviewer 3 Report
The manuscript “Metabolite diversity in pulp segments, skin, leaves and bark of a red-fleshed 'Baya Marisa' apple cultivar” represent rather a lack of novelty. Several studies with red flesh apples have also been published already with one already in the 1992 year (please see papers listed below).
https://doi.org/10.3390/metabo11060378
https://doi.org/10.1016/j.fitote.2012.04.017
https://doi.org/10.1016/j.jff.2014.06.013
https://doi.org/10.1016/0308-8146(92)90223-O
https://doi.org/10.1016/j.scienta.2017.02.041
Additional problem is that authors already published paper where red-fleshed 'Baya Marisa' apple cultivar was compared with the ‘Golden Delicious’ (in the same journal).
https://doi.org/10.3390/horticulturae8090811
This time authors comparing with the leaves and bark of the same cultivar. It is not a good practice.
The phenolic compositions in bark, leaves and different apple parts are well known (please see papers listed below).
https://doi.org/10.1002/jsfa.4093
https://doi.org/10.3390/ph15020244
https://doi.org/10.1016/j.indcrop.2015.05.030
Some technical comments.
- Below the tables where the phenolic compounds were quantified should be listed which kinds of compounds were semi-quantified and which kind of standard was used for it semi-quantification;From scientific point of view the term “peel” is more correct than “skin”;
- Presence of peonidin-3-O-galactoside should be re-checked since in any previous studies was not reported the presence of this anthocyanin in red flesh apples;
- From a mathematical point of view more correct is to express the results as “1076 ± 108” instead of “1075.65 ± 108.18”;
- The results discussion is quite poorly described e.g. “Correspondingly, Juhart et al. [51] reported that scab-susceptible cultivar 'Golden Delicious' contained significantly less total quercetins in skin and pulp compared to 'Baya Marisa' tissues. The content of quercetin-3-O-rhamnoside in 'Baya Marisa' pulp decreased from P1 to P3 but the levels were significantly lower to those measured in the skin.” – what do you mean by “significantly”? This term should be used for statistical results description by providing the “p” value. Without providing information on how many times or % higher or lower, when the results are compared, the provided discussion has a very low informative value. Please be more detailed.
- For Latin names e.g. “Malus domestica” please use italic style;
- “O” in all names of phenolic compounds e.g. quercetin-3-O-rutinoside should be written by italic style;
- Conclusions “The group of flavanols predominates in bark and peel as they play a very important role in plant’s defense, for example against apple scab.”- 1st, this sentence requires reference; 2nd, it cannot be placed in the conclusions part since is not associated with the result of the present study.
non
Author Response
Dear reviewer,
Thank you for your useful comments and suggestions for improvements of our work, particularly al the useful references and links. We have answered your queries and corrected the text accordingly (the added text or altered sentences are marked with tracking tool). The answers to your comments are listed below:
The manuscript “Metabolite diversity in pulp segments, skin, leaves and bark of a red-fleshed 'Baya Marisa' apple cultivar” represent rather a lack of novelty. Several studies with red flesh apples have also been published already with one already in the 1992 year (please see papers listed below).
https://doi.org/10.3390/metabo11060378
https://doi.org/10.1016/j.fitote.2012.04.017
https://doi.org/10.1016/j.jff.2014.06.013
https://doi.org/10.1016/0308-8146(92)90223-O
https://doi.org/10.1016/j.scienta.2017.02.041
Additional problem is that authors already published paper where red-fleshed 'Baya Marisa' apple cultivar was compared with the ‘Golden Delicious’ (in the same journal).
https://doi.org/10.3390/horticulturae8090811
This time authors comparing with the leaves and bark of the same cultivar. It is not a good practice.
The phenolic compositions in bark, leaves and different apple parts are well known (please see papers listed below).
https://doi.org/10.1002/jsfa.4093
https://doi.org/10.3390/ph15020244
https://doi.org/10.1016/j.indcrop.2015.05.030
Some technical comments.
- Below the tables where the phenolic compounds were quantified should be listed which kinds of compounds were semi-quantified and which kind of standard was used for it semi-quantification;From scientific point of view the term “peel” is more correct than “skin”;
In Table 2 the information on identification and quantification of compounds has been presented. It includes the names of all 35 identified compounds and standard compounds used for their quantification. Some compounds that lack standards have been quantified with the use of similar compounds – this is also explained in the Material section (L165-166).
According to the suggestion of the reviewer the text was re-checked and the term skin was replaced with peel. In this aspect, we have also altered the title of the manuscript, footnotes and column names in Tables etc.
- Presence of peonidin-3-O-galactoside should be re-checked since in any previous studies was not reported the presence of this anthocyanin in red flesh apples;
We re-checked the chromatograms of anthocyanins. The smallest peak was indeed determined as peonidin-galactoside, and additionally confirmed based on m/z and MS2 fragmentation. But as we cannot perform additional analyzes on samples, we decided to exclude this compound from the manuscript. A recent study on different apple cultivars also reports the presence of peonidin-3-O-galactoside in skin, but none of the cultivars were red-fleshed https://www.mdpi.com/2311-7524/9/4/475 .
- From a mathematical point of view more correct is to express the results as “1076 ± 108” instead of “1075.65 ± 108.18”;
The expression of the results was corrected in text. Some compounds have been detected in much smaller amounts and omitting decimal digits would greatly limit precision.
- The results discussion is quite poorly described e.g. “Correspondingly, Juhart et al. [51] reported that scab-susceptible cultivar 'Golden Delicious' contained significantly less total quercetins in skin and pulp compared to 'Baya Marisa' tissues. The content of quercetin-3-O-rhamnoside in 'Baya Marisa' pulp decreased from P1 to P3 but the levels were significantly lower to those measured in the skin.” – what do you mean by “significantly”? This term should be used for statistical results description by providing the “p” value. Without providing information on how many times or % higher or lower, when the results are compared, the provided discussion has a very low informative value. Please be more detailed.
Statistical significance was only reported when comparing the content of metabolites among peel and pulp sections. p-value is reported in the footnotes of Table 3 (p < 0.05). Statistical method is explained in L182-183. Statistical comparison of all tissues identified (i.e- comparison of bark, leaves, fruit sections and peel) gives inconsistent information as some compounds are limited to specific tissues or are present in extremely low amounts in other tissues and therefore, we have only compared different fruit segments and peel. Based on the suggestion of the reviewer the discussion was altered so that significance only applies to the results based on statistical comparison. Other comparisons with literature data are only discusses as similar/lower/higher levels of specific metabolites in tissues. Juhart et al. provided statistical significance between 'Golden Delicious' and 'Baya Marisa' skin and pulp and therefore, the term used in the discussion is appropriate. Similarly, the levels of quercetin-3-O-rhamnoside in 'Baya Marisa' skin in our study were significantly higher compared to levels quantified in P1, P2 and P3 (p < 0.05). We have however, added, the % when comparing statistically different results.
- For Latin names e.g. “Malus domestica” please use italic style;
Thank you for pointing out our mistakes. The text has been corrected in several places.
- “O” in all names of phenolic compounds e.g. quercetin-3-O-rutinoside should be written by italic style;
Thank you for pointing out our mistakes. The text has been corrected throughout the manuscript.
- Conclusions “The group of flavanols predominates in bark and peel as they play a very important role in plant’s defense, for example against apple scab.”- 1st, this sentence requires reference; 2nd, it cannot be placed in the conclusions part since is not associated with the result of the present study.
The conclusion was rewritten to better present the relevant results and focuses on demonstrating the practical implications of our findings. No references have been cited in this section.
Round 2
Reviewer 3 Report
The manuscript “Metabolite diversity in pulp segments, peel, leaves, and bark of a red-fleshed 'Baya Marisa' apple cultivar” has been slightly improved.
In my opinion, the manuscript still requires some revision at least.
Several reports could be discussed as well, for instance:
- the phenolic compositions in bark, leaves and different apple parts:
https://doi.org/10.1002/jsfa.4093
https://doi.org/10.3390/ph15020244
https://doi.org/10.1016/j.indcrop.2015.05.030
- some positive health effects of apple phenolics:
https://doi.org/10.1002/jsfa.6779
I still think that not happen the best that authors already published paper where red-fleshed 'Baya Marisa' apple cultivar was compared with the ‘Golden Delicious’ (in the same journal).
https://doi.org/10.3390/horticulturae8090811
Some technical comments.
- From a mathematical point of view the number after the comma does not matter if the error is higher than 1. Please make corrections with all data. For e.g. 8.95 ± 1.41 it should be “9 ± 1”; 22.94 ± 1.78 should be “23 ± 2”; etc.
- The results discussion still requires improvement. In the text still can be found “skin” instead of “peel”.
non
Author Response
Dear reviewer,
Thank you for your comments and suggestions for improvements of our work. We have corrected the text accordingly (the added text or altered sentences are marked with tracking tool). The answers to your comments are listed below:
References
In my opinion, the manuscript still requires some revision at least.
Several reports could be discussed as well, for instance:
- the phenolic compositions in bark, leaves and different apple parts:
https://doi.org/10.1002/jsfa.4093
https://doi.org/10.3390/ph15020244
https://doi.org/10.1016/j.indcrop.2015.05.030
Thank you for the suggested references. The composition of phenolics has been discussed in the initial version of the manuscript but additional references suggested by the reviewer have been included in the revised text. The first reference https://doi.org/10.1002/jsfa.4093 is a study conducted by our research team and reports the pattern of some phenolics in leaves and fruit parts of four apple cultivars (two scab-resistant and two scab-susceptible cultivars) in two years. Another study reporting similar effects has been already cited in the initial text (https://doi: 10.4141/CJPS08202). The composition in both cited references is similar to the recent reports and the differences in levels have been mainly ascribed to stress caused by Venturia infection (higher in susceptible cultivars) or environmental factors. All this has already been thoroughly discussed in the initial text based on many recent references (L316-346 and in all segments of the discussion reporting various classes of phenolics). The main aim of the second reference https://doi.org/10.3390/ph15020244 is methodological (provides optimization of the chromatographic separation conditions) and reports content levels of phloridzin and selected phenolics in leaves, bark, and buds of 13 apple cultivars. The reference has been included in text mainly as it reports the distribution of metabolites in the bark, which has rarely been studied. The last reference https://doi.org/10.1016/j.indcrop.2015.05.030 reports the composition of phenolics in tissues (seeds, stems, fruit with peel and core) of crab apple pomace. It is not comparable to our results as the plant material is different (crab apple may refer to several species of the Malus genus, including M. sylvestris, M. angustifolia and hybrids) and fruit pomace is a by-product of processed juices or other goods and therefore the composition of this material differs considerably from the composition of primary source.
- some positive health effects of apple phenolics:
https://doi.org/10.1002/jsfa.6779
Several references have been included in the Introduction and Discussion reporting the effects of apple phenolics on human health: https://doi: 10.1021/jf020782a, https://doi: 10.1021/acs.jafc.6b02931, https://doi: 10.3945/an.111.000513, https://doi: 10.1016/j.jnutbio.2009.03.008, https://doi: 10.1016/j.arabjc.2021.103188, https://doi: 10.1002/dmrr.532, https://doi: 10.1155/2013/801457, https://doi:10.1111/j.1745-4514.2009.00257. The study endorsed by the reviewer was also included as it reports the effect an unripe apple preparation containing phlorizin on diminishing hyperglycaemic effects in healthy individuals.
I still think that not happen the best that authors already published paper where red-fleshed 'Baya Marisa' apple cultivar was compared with the ‘Golden Delicious’ (in the same journal).
The previously published study on Baya Marisa apples by our research team is an initial report on the composition of metabolites in fruit and peel of this cultivar. It has been published in Horticulturae Journal. This study reports the distribution of phenolics in other tissues as well as in different segments of the Baya Marisa fruit. This cultivar in namely known for its red pulp which varies in intensity from the peel inwards. The results therefore further describe this cultivar and important differences in phenolics in relation to fruit depth.
- From a mathematical point of view the number after the comma does not matter if the error is higher than 1. Please make corrections with all data. For e.g. 8.95 ± 1.41 it should be “9 ± 1”; 22.94 ± 1.78 should be “23 ± 2”; etc.
The expression of the results was corrected where applicable. Some compounds have been detected in much smaller amounts and omitting decimal digits would greatly limit precision. Most references suggested by the reviewer for citing purposes (see comments above) also report the results with similar precision than our study.
The results discussion still requires improvement. In the text still can be found “skin” instead of “peel”.
The discussion was supported by many references and further improved with the ones suggested by the reviewer. The text was re-checked and the term skin was exchanged with peel.
