Genotype and Maturity Stage Affect the Content and Composition of Polyamines in Tomato—Possible Relations to Plant and Human Health

Round 1

Reviewer 1 Report

In the present manuscript, the authors have analysed the polyamine profile of eight tomato genotypes in relation to their maturity stage and quality traits and discuss how this can impact human health and plant development and adaptation.

The study focuses on an interesting topic, such as polyamine sources for human consumption and their implication for health. Here I discuss some points to be addressed before considering this manuscript for publication.

• Line 33. Surprisingly, the authors state that "PAs are widely allocated in the cell's plasma membrane". This is not true. The reference cited addresses the TRANSPORT of polyamines across the plasma membrane (i.e. polyamine fluxes). There are tons of literature regarding polyamine localisation, so please read some and re-write the sentence.
• Line 40. The general abbreviation for reactive oxygen species is "ROS" and not "ROs". Also, please, use a sentence such as "play a role in biotic stress" instead of "antimicrobial activity".
• Polyamines are primarily known to be involved in cell division and are widely classified as growth regulators. That's one of the reasons why polyamine composition varies during the human lifespan. In that regard, it is known for half a century that cancer cells have increased polyamine content and metabolism. Please, discuss how this relates to the supposed benefits of polyamine intake and put it in context.
• Line 78. Studies are not "lacking". As the authors indicate in lines 303-305, there are some studies. If any, one could argue that such studies are "scarce".
• Table 1. The use of a table is unnecessary here. Please write in the Methods text the name of the genotypes used and the seed producer in brackets. The rest of the info is irrelevant. As of 2021, there are more than 100 tomato varieties in the market. Please explain the reason for choosing those 8.
• Line 128. Is this Putrescine dihydrochloride?
• Line 147. Why do you measure ascorbic acid? Explain it briefly somewhere in the text.
• Line 183, polyamine analysis. Please add the reference of the method used other than for the extraction. Explain what fraction of polyamines are you measuring and explain why in the text: Free soluble polyamines? Conjugated?
• Move Table 2 and Figure 1 to supplemental.
• Line 264. What other crops? Add references.
• Visually speaking, the manuscript is flawed. Please, create a panel with pictures of the eight tomato varieties and the different maturation stages, as Figure 1. In each image, also include when (day) the photo was taken. It is important to the reader to know how the maturation process compares between genotypes.
• Table 3. "Quality traits" do not include polyamine content. Please make a clear distinction here and in the text between quality traits and polyamine profile.
• Move Table 3 to supplemental.
• Table 4. Are you comparing the same varieties and maturity stage (which doesn't mean the same days since the timing is also affected by external factors) of tomatoes between studies? If not, this is pointless and should be put into context to explain the differences.
• Move Table 4 to supplemental.
• Figure 2. Use the "PCA" abbreviation and reduce the font size of the different variables in Fig 2A. Align "A" and "B" graph letters. Please use colours instead of letters to indicate the maturity stages in figure 2B.
• Figure 3. Include the Spm results and use a colour code for the different maturity stages. Also, the different letters indicating significant differences are wrongly placed; The different letters are meant to compare the polyamine levels between genotypes, so please correct this.
• It is surprising (to say the least) that in a paper meant to analyse polyamine profiles in different maturity stages, the authors completely ignore, despite the multiple pieces of literature generated over the years, the DIRECT connection between polyamine biosynthesis and ethylene biosynthesis. Explain this in detail in the discussion (also at the genetic level), and discuss how this relates to the data and ideas presented.
• Discuss what external factors used during the growth of the plant (nutrient solution? Light regime? Temperature? Stress? Etc) could affect the polyamine content in the tomato. Could this be a way to increase the polyamine content other than the genotype or the maturity stage?
• Consider an alternative title for the manuscript, such as "Genotype and maturity stage affect the content and composition of polyamines in tomato".

Author Response

Dear reviewer,

We appreciate your valuable comments and suggestions, and your thorough reading and correction of mistakes in the manuscript. Your comments and suggestions have been taken into consideration accordingly, to improve the paper. We have made every attempt to follow your suggestions and recommendations and all corrections are marked in the manuscript by track changes. Below we provide point-by-point responses to your comments.

In the present manuscript, the authors have analysed the polyamine profile of eight tomato genotypes in relation to their maturity stage and quality traits and discuss how this can impact human health and plant development and adaptation.

The study focuses on an interesting topic, such as polyamine sources for human consumption and their implication for health.

Thanks for the positive comments.

Here I discuss some points to be addressed before considering this manuscript for publication.

• Line 33. Surprisingly, the authors state that "PAs are widely allocated in the cell's plasma membrane". This is not true. The reference cited addresses the TRANSPORT of polyamines across the plasma membrane (i.e. polyamine fluxes). There are tons of literature regarding polyamine localisation, so please read some and re-write the sentence.

Thanks for pointing out this mistake, the text has been corrected accordingly.

• Line 40. The general abbreviation for reactive oxygen species is "ROS" and not "ROs". Also, please, use a sentence such as "play a role in biotic stress" instead of "antimicrobial activity".

Changes made accordingly

• Polyamines are primarily known to be involved in cell division and are widely classified as growth regulators. That's one of the reasons why polyamine composition varies during the human lifespan. In that regard, it is known for half a century that cancer cells have increased polyamine content and metabolism. Please, discuss how this relates to the supposed benefits of polyamine intake and put it in context.

Thank you for your suggestions. Discussion and context have been improved accordingly following recommendations from the reviewer.

• Line 78. Studies are not "lacking". As the authors indicate in lines 303-305, there are some studies. If any, one could argue that such studies are "scarce".

The point is well taken and modifications done accordingly.

• Table 1. The use of a table is unnecessary here. Please write in the Methods text the name of the genotypes used and the seed producer in brackets. The rest of the info is irrelevant. As of 2021, there are more than 100 tomato varieties in the market. Please explain the reason for choosing those 8.

Thanks for useful suggestions. Table 1 has been omitted and text improved accordingly.

• Line 128. Is this Putrescine dihydrochloride?
•  

Thanks for the question. We used Putrescine with the ref no 51799-100MG from Sigma-Aldrich which has now been clarified in the text.         

• Line 147. Why do you measure ascorbic acid? Explain it briefly somewhere in the text.

Thanks for your suggestions. The text has been improved accordingly.

• Line 183, polyamine analysis. Please add the reference of the method used other than for the extraction. IExplain what fraction of polyamines are you measuring and explain why in the text: Free soluble polyamines? Conjugated?’

Thanks for your prompt suggestions. We have included literature and explanation accordingly in the materials and methods section.

• Move Table 2 and Figure 1 to supplemental. Just eliminated from the manuscript

Table 2 and Figure 1 were moved to “supplemental material” archive as Table S1 and Figures S1 respectively.

• Line 264. What other crops? Add references

References added accordingly.

• Visually speaking, the manuscript is flawed. Please, create a panel with pictures of the eight tomato varieties and the different maturation stages, as Figure 1. In each image, also include when (day) the photo was taken. It is important to the reader to know how the maturation process compares between genotypes.

A new Figure 1 has been included as per reviewers suggestion.

• Table 3. "Quality traits" do not include polyamine content. Please make a clear distinction here and in the text between quality traits and polyamine profile.

The distinction has been included accordingly.

• Move Table 3 to supplemental.

Table 3 has been moved to supplemental material as Table S2.

• Table 4. Are you comparing the same varieties and maturity stage (which doesn't mean the same days since the timing is also affected by external factors) of tomatoes between studies? If not, this is pointless and should be put into context to explain the differences.

Text that discusses this fact has been included in the results and discussion section.

• Move Table 4 to supplemental.

Table 4 has been moved to supplemental material as Table S3.

• Figure 2. Use the "PCA" abbreviation and reduce the font size of the different variables in Fig 2A. Align "A" and "B" graph letters. Please use colours instead of letters to indicate the maturity stages in figure 2B.

Changes to Figure 2 has been made according to reviewer’s suggestion.

• Figure 3. Include the Spm results and use a colour code for the different maturity stages. Also, the different letters indicating significant differences are wrongly placed; The different letters are meant to compare the polyamine levels between genotypes, so please correct this.

Thanks for useful comments. Changes have been made accordingly and letters are now better explained.

• It is surprising (to say the least) that in a paper meant to analyse polyamine profiles in different maturity stages, the authors completely ignore, despite the multiple pieces of literature generated over the years, the DIRECT connection between polyamine biosynthesis and ethylene biosynthesis. Explain this in detail in the discussion (also at the genetic level), and discuss how this relates to the data and ideas presented.

The connection between polyamine and ethylene biosynthesis is now discussed in the manuscript.

• Discuss what external factors used during the growth of the plant (nutrient solution? Light regime? Temperature? Stress? Etc) could affect the polyamine content in the tomato. Could this be a way to increase the polyamine content other than the genotype or the maturity stage?

A discussion on this topic has been included in the results and discussion section.

• Consider an alternative title for the manuscript, such as "Genotype and maturity stage affect the content and composition of polyamines in tomato".

Thanks for the suggestion. The title has been modified accordingly.

Once again, we thank the reviewer for valuable comments and suggestions and hope that with the changes made, the manuscript is now ready for publication in Horticulturae.

Reviewer 2 Report

The research paper is written nicely, however, there a few are points that need to be clarified as below;

1. Mentioning that "five healthy plants were selected of each genotype for further cultivation", how many replicates were chosen ? deriving data from just five plants is not sufficient.
2. The scientific name of the tomato is not written in italic in many places.
3.  Which human health parameters were assessed in this study?

Author Response

Dear reviewer,

We appreciate your valuable comments and suggestions, and your thorough reading and correction of the manuscript. Your comments and suggestions have been taken into consideration accordingly, to improve the paper. We have made every attempt to follow your suggestions and recommendations and all corrections are marked in the manuscript by track changes. Below we provide point-by-point responses to your comments.

The research paper is written nicely, however, there a few are points that need to be clarified as below;

 Thanks for the positive comment.

1. Mentioning that "five healthy plants were selected of each genotype for further cultivation", how many replicates were chosen ? deriving data from just five plants is not sufficient.

Thanks for pointing out this unclearness, which is now better described in the manuscript.

1. The scientific name of the tomato is not written in italic in many places.

Thanks for pointing out this, which is now corrected.

1.  Which human health parameters were assessed in this study?

Basically no human health parameters were assessed in this study but implications for health parameters are discussed. This discussion has now been improved and clarified.

Once again, we thank the reviewer for valuable comments and suggestions and hope that with the changes made, the manuscript is now ready for publication in Horticulturae.

Reviewer 3 Report

Gutierrez et al., investigated the effect of tomato genotype, maturity stage and their interactions, to the content and composition of Polyamines. This manuscript is well conceptualized and written, however authors need to show some positive correlation between their findings with human health.

1. It is already known that PAs are good for health, here authors should highlight the significance of this study compared to already published works.
2. There are other intake sources for PAs, and why this study is confined to only tomatoes, this needs to be clearly highlighted in the introduction part.

Author Response

Dear reviewer,

We appreciate your valuable comments and suggestions, and your thorough reading and correction of mistakes in the manuscript. Your comments and suggestions have been taken into consideration accordingly, to improve the paper. We have made every attempt to follow your suggestions and recommendations and all corrections are marked in the manuscript by track changes. Below we provide point-by-point responses to your comments.

Gutierrez et al., investigated the effect of tomato genotype, maturity stage and their interactions, to the content and composition of Polyamines. This manuscript is well conceptualized and written,

Thanks for the positive comments.

however authors need to show some positive correlation between their findings with human health.

Thanks for pointing out this. We have extended the discussion about the health benefits of polyamines and the possible impact on health from the present study.

1. It is already known that PAs are good for health, here authors should highlight the significance of this study compared to already published works.

Thanks for the comment. The health part of the manuscript has been extended and further discussed, and also highlights made more clear.

1. There are other intake sources for PAs, and why this study is confined to only tomatoes, this needs to be clearly highlighted in the introduction part.

Thanks for this suggestion. Changes have been done accordingly.

Once again, we thank the reviewer for valuable comments and suggestions and hope that with the changes made, the manuscript is now ready for publication in Horticulturae.

Round 2

Reviewer 1 Report

The authors have addressed the points raised in the previous review. Still, a couple of important issues are to be addressed, mostly affecting the results' representation.

• Line 34-35. I'm afraid I have to disagree with the following statement: "In plants, PAs are regulating growth and stress responses through an action on ion transport across membranes in channels and pumps [6]." The regulation of such biological events are more complex and involves multiple roles at different cellular levels. I suggest removing this sentence and just mention the general processes in plants in which polyamine metabolism is involved. Also, please use "metabolism" instead of biosynthesis and "catalysis" (you mean catabolism, I assume).
• Figure 1. If the authors cannot provide a representative number of days for each genotype's maturation process, please remove the date of the picture and modify the legend accordingly. This info is rather confusing and does not provide information on a tomato's maturation process, which was my point. In other words, my point is meant to know if there is any tomato genotype that maturates faster than others when plants are growing together since this can be related to the ethylene-polyamine antagonism.
• Figure 2B. I believe there is a misunderstanding of my point regarding the combination of colours and symbols. My point is meant to simplify the readout of the data. In this sense, I suggested using the symbols of the different varieties in different colours (colours corresponding to the stages). For example, "RG green" will be a green triangle or "HT36 light red" will be a red cross. Please use this way to show the data. Also, make sure that the size of the symbol is big enough to be identified without overlapping excessively.
• Figure 3. The letters are still wrongly used. In each panel, each letter has to serve as a comparison not only for stages within the same genotype but ALSO between genotypes. For example, Tpa levels in Huichol green are different than Tpa levels in HT36 red. Thus, it cannot be "a" in both cases since the difference between both levels is significant. In other words, the letters have to provide the readout of a paired t-test (or similar) comparing EVERYTHING within the same panel. Also, please remove the grey line from the border of each panel, use the same "ug" symbol for Spm as in Spd and Put, and remove the space in "HT 36".

Author Response

Dear reviewer,

We appreciate your valuable comments and suggestions, and your thorough reading and correction of mistakes in the manuscript. Your comments and suggestions have been taken into consideration accordingly, to improve the paper. We have made every attempt to follow your suggestions and recommendations and all corrections are marked in the manuscript by track changes. Below we provide point-by-point responses to your comments.

In the present manuscript, the authors have analysed the polyamine profile of eight tomato genotypes in relation to their maturity stage and quality traits and discuss how this can impact human health and plant development and adaptation.

The authors have addressed the points raised in the previous review.

Thanks for the positive comments.

Still, a couple of important issues are to be addressed, mostly affecting the results' representation.

As you can see from the new version of the manuscript, the suggested issues have been addressed.

Line 34-35. I'm afraid I have to disagree with the following statement: "In plants, PAs are regulating growth and stress responses through an action on ion transport across membranes in channels and pumps [6]." The regulation of such biological events are more complex and involves multiple roles at different cellular levels. I suggest removing this sentence and just mention the general processes in plants in which polyamine metabolism is involved. Also, please use "metabolism" instead of biosynthesis and "catalysis" (you mean catabolism, I assume).

Sentence and reference has been removed as suggested.

Figure 1. If the authors cannot provide a representative number of days for each genotype's maturation process, please remove the date of the picture and modify the legend accordingly. This info is rather confusing and does not provide information on a tomato's maturation process, which was my point. In other words, my point is meant to know if there is any tomato genotype that maturates faster than others when plants are growing together since this can be related to the ethylene-polyamine antagonism.

Changes made accordingly.

Figure 2B. I believe there is a misunderstanding of my point regarding the combination of colours and symbols. My point is meant to simplify the readout of the data. In this sense, I suggested using the symbols of the different varieties in different colours (colours corresponding to the stages). For example, "RG green" will be a green triangle or "HT36 light red" will be a red cross. Please use this way to show the data. Also, make sure that the size of the symbol is big enough to be identified without overlapping excessively.

Thanks for the clarification. In the present version, a figure as the one suggested by the reviewer has been included as Fig S3, and then mean values have been used in Fig 2B to ease for the reader.

Figure 3. The letters are still wrongly used. In each panel, each letter has to serve as a comparison not only for stages within the same genotype but ALSO between genotypes. For example, Tpa levels in Huichol green are different than Tpa levels in HT36 red. Thus, it cannot be "a" in both cases since the difference between both levels is significant. In other words, the letters have to provide the readout of a paired t-test (or similar) comparing EVERYTHING within the same panel. Also, please remove the grey line from the border of each panel, use the same "ug" symbol for Spm as in Spd and Put, and remove the space in "HT 36".

Thanks for the clarification. Including letters from the comparison of all genotypes and stages will make the figure very messy and in fact impossible to read. However, the information of such an analyses might be of interest as the reviewer is pointing out and therefore, the result of such an analyses is added as Table S4. Other suggestions of changes have been done accordingly. As to the comparisons of stages for each of the genotypes, this kind of comparisons are often seen in papers and if one only clearly states what kind of comparisons that are carried out it is normally seen as OK. If the reviewer so prefers, we can take away the letters in the fig and just keep standard deviations.

Once again, we thank the reviewer for valuable comments and suggestions and hope that with the changes made, the manuscript is now ready for publication in Horticulturae.