Effects of Salt Stress on Fruit Antioxidant Capacity of Wild (Solanum chilense) and Domesticated (Solanum lycopersicum var. cerasiforme) Tomatoes

Round 1

Reviewer 1 Report

The Introduction part may be shortened. Namely, some statements and citations are not strongly related to the topic being investigated or they were not connected to the sentences/paragraph preceding and/or following and thus can be deleted. For example, first and second paragraphs deals with totally different topics and thus the reading flow is corrupted. 

The Material and Methods part must be improved in the first part where vegetation trial is being described. Some parts are not clear. For example, what is meant by this description: "Seedlings were grown in growth chambers consisting in four outdoor containers filled with a vermiculite-perlite substrate". What is an outdoor container? Further, it is maybe more appropriate to say rockwool slab instead rock wool bed.

Authors should consider using the term salt-stress instead salinity in some parts of their manuscript.

The Discussion part can be improved by adding a few key references to the results being discussed. Currently, some results were not appropriately discussed.

Author Response

Dear Reviewer 1:

Here are our responses:

1.- The Introduction part may be shortened. Namely, some statements and citations are not strongly related to the topic being investigated or they were not connected to the sentences/paragraph preceding and/or following and thus can be deleted. For example, first and second paragraphs deals with totally different topics and thus the reading flow is corrupted. 

Response of the authors. We do fully agree with the reviewer. According ti his/her suggestion, we removed the second paragraph of the former introduction, which was indeed “out of scope” in order to focus on the salt impact on tomato. The genetic basis of salinity resistance, which was described in this second paragraph, is now briefly mentioned later in the introduction. We also re-organized some sentences in order to more appropriately deal with fruit metabolism. We do believe that this modification will clarify the presentation.

2.- The Material and Methods part must be improved in the first part where vegetation trial is being described. Some parts are not clear. For example, what is meant by this description: "Seedlings were grown in growth chambers consisting in four outdoor containers filled with a vermiculite-perlite substrate". What is an outdoor container? Further, it is maybe more appropriate to say rockwool slab instead rock wool bed.

Response of the authors. We do agree that the former presentation was a bit confusing and misleading. The terms “outdoor” containers refer mainly to tanks containing vermiculite/perlite and that were placed for a short period under portable greenhouses with only semi-controlled environmental conditions. This period was rather short (it occurs just after germination step); Three-weeks old plants were then transferred to the final hydroponic system in fully-controlled greenhouses. We clarify these initial steps of plant culture in the revised version.

3.- Authors should consider using the term salt-stress instead salinity in some parts of their manuscript.

Response of the authors. We do agree with the referee and replace “salinity” by “salt-stress” in some parts of the text where it was justified, but not everywhere. Indeed, from a conceptual point of view, the term “stress” refers to the range of physiological modifications (and symptoms) induced by deleterious environmental conditions. Hence “salt-stress” concerns the physiological modification induced by accumulation of salt ions and modification of plant water status, and it is therefore possible, for example, to mention “salt-stress” resistance instead of “salinity” resistance. Contrary to internal physiological modification, “salinity” refers to the external constraint itself. Accordingly, a value given using electrical conductivity units is an estimation of salinity, but not an estimation of salt stress. We therefore modified the manuscript considering this distinction.

4.- The Discussion part can be improved by adding a few key references to the results being discussed. Currently, some results were not appropriately discussed.

Response of the authors. We do agree with the referee and we made a special effort to clarify discuss, add some new references and improve this section. We refer to the recent work of Zhang et al. (2020) Environ. Exp. Bot. 177, 104145, which provides nice information regarding the role of N-glycosylation processes in the control of sugar metabolism under salt stress conditions. We also integrate findings published by Abdelgawad et al. (2019) in Agronomy 9, 51: this study especially focusses on ascorbic acid content in cherry tomato and it establishes a clear link between ascorbate oxidase activity on the one hand and ascorbate content (and to a lower extent carotene and lycopene concentrations). The work of Sumalan et al. (2020, Agronomy, 10, 500) is also integrated in the discussion since these authors assessed the performance of twenty halotolerant landraces of cherry tomato. They provide new insight regarding the link between salinity resistance and lycopene concentration and this study is interesting to integrate in our discussion. Rodriguez-Ortega et al. (2019; Sci. Rep. 9: 6733) presented a global approach on agricultural and physiological responses of tomato plants grown in different soilless culture systems with saline water under greenhouse conditions. They noticed an important increase in titratable acidity and this work allowed us to deepen the discussion regarding the interest of the high titratable acidity recorded in S. chilense, reinforcing the interest of this species for further breeding approach with domesticated tomato.

Reviewer 2 Report

The subject of the manuscript entitled “Effects of salt stress on fruit antioxidant capacity of wild (Solanum chilense) and domesticated (Solanum lycopersicum var. cerasiforme) tomatoes” totally fits the profile of Agronomy journal. The study delivers some interesting results and can be a source of valuable information. However, the authors made shortcomings that should be corrected and/or revised before the publication of this work. 

1. In key words, the same words as in the title should not be used.
2. The authors should introduce and discuss important mechanisms at the cellular, membrane level, the role of aquaporins in water uptake, the role of osmolytes, as well as Na⁺ and K⁺ accumulation during salt stress. The antioxidant system, both enzymatic and non-enzymatic antioxidants, also should be introduced.
3. Line 49, how did authors define good or bad quality water? High-quality drinking water lacks microelements which are important for plant growth.
4. Line 73, “old local varieties” = landrace?
5. Line 78-80, this is common sense, but authors should cite some paper her.
6. Line 85-87, all previous studies (ref. 27-31) are self-cited here. Self-citation is fine, but only ref. 29 was cited in the discussion. So, ref 27, 28, 30, 31 are unnecessary self-cited?
7. The treatment “0 mM NaCl” should be labeled as control (CK) or blank in M&M, and every table & figure in this manuscript.
8. “cherry tomato” and “wild tomato” in the legend of every figure should be labeled as S. lycopersicum and S. chilense, respectively.
9. In figure 1, the units of FFW and FDW should be “g per fruit”, and the unit of TA should be double-checked.
10. In table 1, the units of all parameters should be corrected as “mg g^-1FW”, “μmol g^-1FW”, and “nmol g^-1FW”, respectively.
11. In figure 2, the unit of total polyphenols should be revised as “μmol gallic acid equivalents g^-1FW”
12. In figure 4, the units of APX and SOD activities should be checked.
13. In figure 5, a footnote is needed to explain the abbreviations of each vector. According to fig 1, SDW and SFW should be revised as FDW and FFW, respectively. Furthermore, the cumulative percentage of CPI and CP2 are 40.12 and 15.64%, respectively. But axis 1 and 2 represented 29.68 and 16.64%, respectively, in line 314-316. In addition, there are 7 plots for G1, 4 plots for G2, and a totally of 11 plots in figure 5B. But there should be 36 samples (2 species * 3 NaCl levels * 6 replicates) in this study.
14. Line 317-318, authors described that this separated 2 species of tomato based on SOD and MDA. However, PROL, TSS, TSS/TA, FDW, FFW, b-CAR, LYC, FRAP, GSHT, ASA, TA, GSH, and PT should be also good to separate 2 species according to Fig 5A.
15. Line 331-335, how did authors take these results together and then knew S. chilense had a higher ability to cope with salinity and behaved as a halophyte? Authors should provide more information and inference according to previous studies which might support the author's hypothesis.
16. Line 358, how did authors define “paramount importance in human diet”? There should be some studies to support the authors’ opinion.
17. Line 363-365, authors should cite some papers.
18. The following research review may help to improve the introduction.
    1. https://doi.org/10.1007/s12033-012-9538-3
    2. https://doi.org/10.3390/agriculture9100223

Author Response

Dear Reviewer 2,

Here are our responses:

1. In key words, the same words as in the title should not be used.

Response of the authors. We do agree with the referee. The present keywords are not included in the title.

2. The authors should introduce and discuss important mechanisms at the cellular, membrane level, the role of aquaporins in water uptake, the role of osmolytes, as well as Na⁺and K⁺ accumulation during salt stress. The antioxidant system, both enzymatic and non-enzymatic antioxidants, also should be introduced.

Response of the authors. We do agree with the referee regarding the fact that the mentioned properties are important key properties influencing the behaviour of the plants exposed to salt stress. This is extremely important during plant growth, at both shoots and roots levels. However, the present paper is not dealing with the vegetative phase of plant growth but rather focuses on fruit properties. Fruit formation is the result of a maturation process implying at the end of the ripening stage a senescence process. These properties are mainly dealing with the antioxidant system, mainly because it is directly involved in plant response, and also because it has important consequences on nutritional properties of the fruits for human consumption. The physiological properties mentioned by the referee were already considered in previous papers from our teams (see Martinez et al., 2012 ref.n°12 and Martinez et al., 2014; ref n°31) and other works (ref 27, 28, 30). These parameters were not again considered in the present manuscript dealing with the same material. As a consequence, it would be extremely confusing for the reader to have an introduction focusing on parameters which are not quantified, and it will be impossible to discuss parameters which were not quantified! We refer to the suggestion of the other reviewer who suggest us to really focus on the precise subject of the article, in order to provide to the reader an adequate take-home message.

3. Line 49, how did authors define good or bad quality water? High-quality drinking water lacks microelements which are important for plant growth.

Response of the authors. This sentence was present in the former version but the paragraph is deleted in the present version. To our point of view, in relation to salinity, “poor quality” water refers to water with a high EC values and even with some traces of organic and inorganic pollutants.

4. Line 73, “old local varieties” = landrace?

Response of the authors. Yes, we can use the term landrace and we made the change according to the suggestion of the referee.

5. Line 78-80, this is common sense, but authors should cite some paper her.

Response of the authors. We do agree and the best citation is the nice review of Flowers and Yeo (Flowers, T.; Yeo, A.R. Breeding for salinity resistance in crop plants: where next? Austr. J. Plant Physiol. 1995, 22, 875-884. doi: 10.1071/PP9950875). It Is now mentioned In the revised version.

6. Line 85-87, all previous studies (ref. 27-31) are self-cited here. Self-citation is fine, but only ref. 29 was cited in the discussion. So, ref 27, 28, 30, 31 are unnecessary self-cited?

Response of the authors. We do agree with the reviewer that self-citation is always a posible prolem; indeed, it might sometimes appear as an attempt of the author to indirectly increase their own citation index, which is not fully honest. For this reason, and because we are honest scientists, we usually avoid self-citation or reduce them a minimum. In the present case, however, the situation is different: Solanum chilense is an interesting halophyte plant species but surprisingly is rarely considered for salinity resistance (it is exhaustively used to study disease resistance). Studies were performed on low temperature (ref 25) or drought (ref 2§): they are both mentioned. BUT, the only studies focusing to mechanisms of salinity resistance in this species were performed by our teams and these studies precisely considered most of the properties listed by the referee as important properties involved in salinity resistance. We thus mention these studies because it is important for understanding the present manuscript and because no other studies are available in the literature.

7. The treatment “0 mM NaCl” should be labeled as control (CK) or blank in M&M, and every table & figure in this manuscript.

Response of the authors. We do agree with the referee and this has been corrected in M&M, and every table and figure in the manuscript.

8. “cherry tomato” and “wild tomato” in the legend of every figure should be labeled as S. lycopersicumand  chilense, respectively.

Response of the authors. We do agree with the referee and the legend of every figure was labeled as S. lycopersicum and S. chilense.

9. In figure 1, the units of FFW and FDW should be “g per fruit”, and the unit of TA should be double-checked.

Response of the authors. We do agree with the reviewer and this was corrected and checked.

10. In table 1, the units of all parameters should be corrected as “mg g^-1FW”, “μmol g^-1FW”, and “nmol g^-1FW”, respectively.

      Response of the authors. We do agree with the referee and this has been corrected.

11. In figure 2, the unit of total polyphenols should be revised as “μmol gallic acid equivalents g^-1FW”.

Response of the authors. We do agree with the referee and this has been corrected.

12. In figure 4, the units of APX and SOD activities should be checked.

Response of the authors. We do agree with the referee and the units of APX and SOD activities were checked.

13. In figure 5, a footnote is needed to explain the abbreviations of each vector. According to fig 1, SDW and SFW should be revised as FDW and FFW, respectively. Furthermore, the cumulative percentage of CPI and CP2 are 40.12 and 15.64%, respectively. But axis 1 and 2 represented 29.68 and 16.64%, respectively, in line 314-316. In addition, there are 7 plots for G1, 4 plots for G2, and a totally of 11 plots in figure 5B. But there should be 36 samples (2 species * 3 NaCl levels * 6 replicates) in this study.

Response of the authors. We do agree with the referee. The abbreviations of each vector were explained in a footnote. SDW and SFW were changed by FDW and FFW. Figure 5 A and B were improved and corrected according the reviewer and the cumulative percentage of CP1 and CP2 were checked in the figure 5 A and 5 B and in the manuscript. In this work were analyze 36 samples (2 species * 3 NaCl levels * 6 replicates) and placed in the 36 plots in the figure 5B.

14. Line 317-318, authors described that this separated 2 species of tomato based on SOD and MDA. However, PROL, TSS, TSS/TA, FDW, FFW, b-CAR, LYC, FRAP, GSHT, ASA, TA, GSH, and PT should be also good to separate 2 species according to Fig 5A.

Response of the authors. We do partially agree with the referee. We have preferred to highlight the separation of the two species  in Fig 5B. These results were corrected and checked.

15. Line 331-335, how did authors take these results together and then knew S. chilense had a higher ability to cope with salinity and behaved as a halophyte? Authors should provide more information and inference according to previous studies which might support the author's hypothesis.

Response of the authors. We slightly modified the sentence in order to better discriminate what are the causes and what are the consequences. We believe that true halophyte was mainly selected in salt-containing environment. As a consequence, they constitutively produce suitable compounds to cope with salinity (in the present case, some antioxidant content). This “constitutive” adaptation allows the plant to react very quickly to any rise of salinity and does not require additional metabolic costs for the synthesis of the required compound. The situation for glycophyte is different: during evolution, they were not exposed frequently to salt and thus do not express these properties in a constitutive way. They may have the opportunity to induce the response when it is justified: triggering the suitable response will take time (for salt sensing, signal transduction, gene expression, protein synthesis and maturation) and will require energy (in some cases at the expense of growth). The fact that S. chilense maintained under control conditions exhibit high antioxidant capacity is an indication (although not a proof), that it behaves as a real halophyte. Some references which discuss those aspects are provided in the revised version and we change the sentence to be less affirmative.

16. Line 358, how did authors define “paramount importance in human diet”? There should be some studies to support the authors’ opinion.

Response of the authors. The fact that vitamin C is an extremely important compound for human health, and thus in human diet, is so evident and so well established that we found unnecessary to mention reference for such an obvious affirmation. However, we added in the revised version references that may support this reality in the specific case of tomato.

17. The following research review may help to improve the introduction.
    10. https://doi.org/10.1007/s12033-012-9538-3
    11. https://doi.org/10.3390/agriculture9100223

Response of the authors. We do thank the reviewer for these two interesting references. They contain a large set of quite interesting data and we Will for sur keep this precios material for further use. However, in the present case, we do not really understand how they can be included in the Introduction since none of them is dealing with fruit. The first reference (a) is an complete review of the molecular basis of signalling in relation to gene regulation and transduction pathways. It provides full information regarding transcription factors: but these molecular aspects are not considered in our study (see response of the authors to comment n°1). The second reference is dealing with sugar beet roots. The only common point between sugar beet roots and tomato fruits is that they are both sink organs! It is well established, however, that modalities of development are completely different in the two systems (and even mechanisms of phloem unloading are completely different). Mauring fruit is a senescing organ…. while sugar beet root is astoring organ accumulating sucrose during the first year of this bianual plant. We really do not see any common points which could justify include this reference in the present introduction.