Efficacy and Comparison of Different Strategies for Selenium Biofortification of Tomatoes

Round 1

Reviewer 1 Report

In the manuscript”Efficacy and comparison of different strategies for selenium biofortification of tomatoes « authors Anton Shiriaev, Beatrice Pezzarossa, Irene Rosellini, Fernando Malorgio, Silvia Lampis, Antonio Ippolito, and Pietro Tonutti, compared the efficacy, in terms of Se accumulation in different tomato plant organs, among the two Se forms: sodium selenate and SeNPs.

 

The manuscript of Shiriaev et al. assessed the effectiveness of on-plant enrichment (whole-plant spraying) and designed possible alternative strategies for postharvest fruit enrichment.

 

Abstract

I suggest adding one sentence after last sentence in the abstract, since it is not clear enough.

OK

 

Key words Are OK.

 

Introduction

Clear with the relevant references cited.

 

Materials and Methods

 

OK

 

Results and Discussion

Too long part in the beginning of the section “ Results and Discussion” without references.

 

L 264-266 Where is the year in the following sentence? “However, Zhu et al. tested a different tomato cultivar from ours (cv. Provence), which might potentially have a different Se absorption capacity. This consequently makes  interpretating the results less straightforward. »

 

 I miss some more discussion with the literature data and information about translocation of Se in plants in terms of phloem and xylem.

 

Is informative, concise, with the appropriate literature cited.

 

Specific comments

I recommend in the future, also to measure some morphological parameters of tomato.

 

The idea of MS is interesting, and the results are clearly presented. I want to highlight the clear and comprehensive work of the author. I found the manuscript well-written and very clear.

 

My suggestions: minor revision

Author Response

The manuscript of Shiriaev et al. assessed the effectiveness of on-plant enrichment (whole-plant spraying) and designed possible alternative strategies for postharvest fruit enrichment. 

Abstract

OK

I suggest adding one sentence after last sentence in the abstract, since it is not clear enough.

A: A sentence has been  added (please note that that the new text in the manuscrip is in red characters)

Key words Are OK.

Introduction

Clear with the relevant references cited.

Materials and Methods

OK

Results and Discussion

Too long part in the beginning of the section “Results and Discussion” without references.

It is not clear what is the “beginning of the section” the Reviewer refers to. We started (line 203) with a reference regarding the use of NP and then we described our results (with no need to cite any published literature). Then (line 240 and following,  but this is not the beginning  of the section any more) we proceed with some discussion of the obtained results citing several references.

L 264-266 Where is the year in the following sentence? “However, Zhu et al. tested a different tomato cultivar from ours (cv. Provence), which might potentially have a different Se absorption capacity. This consequently makes  interpretating the results less straightforward. »

The Reviewer is right, we forgot to add the numerical order in the Reference list of the specific citation .

I miss some more discussion with the literature data and information about translocation of Se in plants in terms of phloem and xylem.

According to the Reviewer’s suggestion we have added specific sentences concerning translocation  hypotheses of Se in the context of our results and experimental plan in the Conclusion session.

Is informative, concise, with the appropriate literature cited.

Specific comments

I recommend in the future, also to measure some morphological parameters of tomato.

Thanks for the suggestion. We agree that this  will be beneficial in future experiments on the topic.

.The idea of MS is interesting, and the results are clearly presented. I want to highlight the clear and comprehensive work of the author. I found the manuscript well-written and very clear.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors presented the manuscript on the enrichment of generative and vegetative organs of tomatoes with one of the most important trace elements - selenium. However, the planned use for the production of dietary supplements of the leaves and stems of the plant belonging to the Solanaceae family, which contain various alkaloids, is of concern.

In the Introduction, the authors talk about the broad therapeutic (protective) effect of selenium compounds. Information should also be provided by what mechanisms such action is provided.

Notes on the manuscript:

Line 14. "Right concentrations" - not quite a scientific term, it needs to be replaced.

Line 15. In many countries, the tomato is classified as a vegetable, not a fruit.

Lines 78-79. Is there any literature confirmation of possible environmental pollution by the doses of selenium used?

Line 83. Previously, the authors write that organic selenium compounds are the most optimal for the human health. Selenium with an oxidation state of 0 is chemically inactive. Thus, it does not form such compjnds well.

Lines 87-88. Literature references are required.

Line 119. It is necessary to describe in more detail the synthesis and structure of the resulting selenium nanoparticles, especially since the amino acid is also present there.

The experiment described in section 2.1.1. and 3.1 serves exclusively to choose the working concentrations and is of little interest.

Line 237. If the results do not differ statistically (significant), then it is impossible to talk about efficiency at all.

Author Response

The authors presented the manuscript on the enrichment of generative and vegetative organs of tomatoes with one of the most important trace elements - selenium. However, the planned use for the production of dietary supplements of the leaves and stems of the plant belonging to the Solanaceae family, which contain various alkaloids, is of concern.

A: As clearly stated , our main interest was to enrich tomato fruit. Nevertheless we had also report our findings of vegetative organs to model the pattern of Se distribution after the whole-plant spraying. Indeed, the conclusion about improved nutraceutical value is only applied for fruit, the edible part of tomato plant.

In the Introduction, the authors talk about the broad therapeutic (protective) effect of selenium compounds. Information should also be provided by what mechanisms such action is provided.

The authors have an expertise in the various field of plant science and agronomy. What suggested by the Reviewer goes beyond the goal of the paper and our expertise.

Notes on the manuscript:

Line 14. "Right concentrations" - not quite a scientific term, it needs to be replaced.

Thank you, the word “right” was replaced with “appropriate”

Line 15. In many countries, the tomato is classified as a vegetable, not a fruit.

The goal of our trials was to evaluate the effect of different Se-enrichment protocols in enriching the edible part of tomato plant, that , botanically speaking, is a fruit (berry).

Lines 78-79. Is there any literature confirmation of possible environmental pollution by the doses of selenium used?

Yes, two references are reported on  lines  80 and 81. However, it is difficult to discuss these data in the context of the doses applied in our experiment, as far as the technology we proposed was not yet practically applied .

Line 83. Previously, the authors write that organic selenium compounds are the most optimal for the human health. Selenium with an oxidation state of 0 is chemically inactive. Thus, it does not form such compjnds well.

Based on the literature (Wang et al. [32] and Neysanian et al [35]) Se⁰  appears to be more readily assimilated to organic forms in rice and tomatoes, compared with Se (VI).  We improved the paragraph below , reporting this information after pointing out the innovative options of using nanoparticles for delivering  Se⁰

Lines 87-88. Literature references are required.

Done

Line 119. It is necessary to describe in more detail the synthesis and structure of the resulting selenium nanoparticles, especially since the amino acid is also present there.

According to the Reviewer’ comment we better detailed (also updating the citation regarding the synthesis procedure) the NP features.

The experiment described in section 2.1.1. and 3.1 serves exclusively to choose the working concentrations and is of little interest.

We politely disagree with the Reviewer. Besides pointing out the genotype effect of   (with marked difference in terms of fruit phenotype), section 2.1.1. shows the importance of the developmental stage in correspondence of which the treatments are performed, reporting important effects in terms of Se concentration and distribution  in leaves and fruit following a treatments performed at blooming (the treatment on MicroTom was performed at IG stage of the fruit)

Line 237. If the results do not differ statistically (significant), then it is impossible to talk about efficiency at all.

Thanks for the pertinent comment. We made a mistake in reporting the concentrations and the significant difference in the roots. Now the text has been corrected. In addition, and according to the appropriate Reviewer’ comment,  we also re-formulated the sentence and the comments concerning the fruit concentrations.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

It should be added to the text that only tomato berries can be used for the production of nutritional supplements. Is it possible to replace the spraying of tomatoes with the addition of known doses of selenium compounds in the manufacture of nutritional supplements? It will be cheaper and more efficient. In addition, the question remains unclear to me why selenium in the oxidation state 0 has a greater chemical affinity for organic substances. I would like to see a scientific explanation, and not a statement of facts in two papers.

Author Response

We thank the Reviewer for his/her comments that allow us to clarify some aspect of our manuscript.

C: It should be added to the text that only tomato berries can be used for the production of nutritional supplements.

A:  In the second line of the introduction we already stated that, as alternative to nutritional/food supplements (products that are usually ingested in capsule, tablet or liquid form),  biofortification represents an attractive option. Hence and for this purpose,  only the biofortification of edible parts of the plant is of interest: and the edible part of a tomato plant is the fruit (berry). Other crops may have different targets than the fruit, depending on the edible part : tubers in potato, roots in carrot (both already present in the market). Besides the title (that already reports “tomatoes” as object of our technical/scientific interest),  we have emphasized/reinforced this aspect by adding in line 98-98 a specific sentence.

C: Is it possible to replace the spraying of tomatoes with the addition of known doses of selenium compounds in the manufacture of nutritional supplements? It will be cheaper and more efficient.

A: 

In many Se‐enriched plants most Se is in the major organic form (selenomethionine, selenocysteine and methylselenocysteine) that is more available to humans and more efficient in increasing the selenium content, especially in the blood, than the inorganic forms. The consumption of Se‐enriched plants thus appears to be more beneficial than nutritional supplements. The overall production of Se supplements urgently needs control because suppliers provide information on total Se concentration, but little or no information on the Se species is present. Furthermore,  supplements are relatively expensive and only a small proportion of the population are likely to take such personal intervention measures, also considering that recent EU
legislation restricts the sale of such supplements

C: In addition, the question remains unclear to me why selenium in the oxidation state 0 has a greater chemical affinity for organic substances. I would like to see a scientific explanation, and not a statement of facts in two papers.

A: The selenium present in the SeNPs is in its elemental form (we are not aware of chemically or biologically synthesized NPs with different form of Se). As reported in line  84,   the use of Se⁰ may represent an advantage in biofortification protocols since it does not interfere as much with cellular processes compared to other forms, and reduces the  risks of environmental pollution and toxicity if compared with treatments with selenate and , in particular, with selenite.

In addition, and this might be the second advantage of Se⁰ , it has been demonstrated and reported in  scientific papers (this was not a goal of our trials)  that  Se⁰ has a more rapid assimilation (not affinity) into organic form such as SeMet and SeCys, when compared to selenate (Se – VI). And this is what we have reported in lines 92-93 mentioning what  was observed previously in rice plants exposed to SeNPs, selenite and selenite (Wang, K. et al.  Translocation and Biotransformation of Selenium Nanoparticles in Rice Seedlings (Oryza Sativa L.). J. Nanobiotechnology 2020, 18, 1–15, doi:10.1186/s12951-020-00659-6). In that case, Authors observed a higher percentage of Se amino acids in plants exposed to SeNPs than in plants exposed to Se (VI).  This could be due to the fact that Se⁰ carried by the NPs is rapidly converted in selenite that in turn can be transformed into Se organic forms. In fact, if selenate is taken up by plants , it must be activated before reduction to selenite that is converted  in selenide  which is incorporated to SeCys. Selenate activation is a rate-limiting step mediated by the ATP sulphurase enzyme  which couples selenate to ATP to generate APSe. APSe is then reduced to selenite via transfer of two electrons from glutathione. (Pilon-Smits et al. 2009 , Curr Opinion in Plant Biol 12(3):267-274 ). The use of Se⁰  would allow to bypass the  slow and energy-consuming process of selenate transformation in selenite.

Since the goal of our paper was limited to a comparative  evaluation  of different biofortification methods in terms of total Selenium accumulation in tomato fruit (and other non-edible organs in the case of spraying trials), and no Se speciation analyses have been carried out,  we do think that a detailed explanation of these physiological mechanisms is not necessary in the context of the manuscript.

 

Author Response File: Author Response.docx