SlSPS, a Sucrose Phosphate Synthase Gene, Mediates Plant Growth and Thermotolerance in Tomato

Round 1

Reviewer 1 Report

The study on tomato heat tolerance by SPS genes is an interesting study and can improve the yield of tomatoes of a specific cultivar. 

I have a few comments on your study which should be answered and should revise in your manuscript.

Methodology on how tomato seeds were purchased, why was Micro-Tom cultivar selected whether this cultivar is a heat-tolerant or heat-sensitive 

The information on how heat stress was given and what time and how many days is missing 

Sperate your conclusion section from the discussion and it should well define your summary of your results and what gaps you have filled and how it can be used in field levels. Your conclusion is very poor.

Your results on root length and fruit weight seem to be odd. Because the plants presented is very small how was fruit weight checked

Can provide the data of flower count also, and how many flowers were aborted and how many changed into flowers.

Whether the crops were grown in greenhouse conditions after transgenic lines were made or in a growth chamber. All information should be provided.

Minor comments

Please check carefully typos and minor errors throughout your MS as I can see at several places 

Please mention full growth conditions since first time the seeds were germinated in greenhouse conditions. 

Please mention what different time periods were selected for sampling

After the construction of constructs, how was genetic transformation done you have not mentioned in your methodology. The whole methodology should be presented. Besides, did you use co-infiltration method please mention. 

The results are not properly explained like how transgenic lines worked better compared to wild type.

I could not see any difference in H2O2 localization (DAB staining) preferably you can reanalyze it

Correct the units of all bar diagrams like SOD, CAT, MDA etc.

Author Response

Dear Reviewer,

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft. We have added necessary information to supplement our results and edited our article extensively. The detailed corrections are listed below.

1. on how tomato seeds were purchased, why was Micro-Tom cultivar selected whether this cultivar is a heat-tolerant or heat-sensitive

We thank for your great comment. As an effective model system for the study of plant biology, Micro-Tom has been widely applied on heat stress research and heat stress negatively impacts growth and development of Micro-Tom cultivar. We have added the reference for choosing Micro-Tom as [27].

Parrotta L, Aloisi I, Faleri C, Romi M, Del Duca S, Cai G. Chronic heat stress affects the photosynthetic apparatus of Solanum lycopersicum L. cv Micro-Tom Plant Physiol Biochem. 2020. 154:463-475.  doi: 10.1016/j.plaphy.2020.06.047. 

2. The information on how heat stress was given and what time and how many days is missing

We thank for your suggestion. We added these information in our revised version to help readers understanding our text.

3. Seperate your conclusion section from the discussion and it should well define your summary of your results and what gaps you have filled and how it can be used in field levels. Your conclusion is very poor.

We appreciate your comments. What gaps we have filled and how it can be used in field levels are truly worthy to discuss. We supplement this part in discussion and modified our conclusion.

4. Your results on root length and fruit weight seem to be odd. Because the plants presented is very small how was fruit weight checked

Thanks. We performed several analyses on multiple phenotypes. Among these phenotypes, root length and fruit weight exhibited significant difference. For the test of root length, seeds of Micro-Tom, over-expression lines (OE) and knock-out lines (CR) were sterilized and sown on ½ MS medium under 18-h light (25 °C)/6-h dark (18 °C) cycle conditions. At least 20 seeds were used and the lengths of primary roots were measured at 2 weeks. Although Micro-Tom is the smallest tomato variety, the plants can obtain enough fruits and seeds for genetic research. Liu et al., researched the function of SlGRAS4 under cold stress using Solanum lycopersicum L. cv. Micro-Tom, including tomato fruits. Therefore the measurement of fruit weight is scientific and reasonable

Liu Y, Shi Y, Zhu N, Zhong S, Bouzayen M, Li Z. SlGRAS4 mediates a novel regulatory pathway promoting chilling tolerance in tomato. Plant Biotechnol J. 2020 Jul;18(7):1620-1633.

5. Can provide the data of flower count also, and how many flowers were aborted and how many changed into flowers.

We thank for your great suggestion. Next step we would observe and conduct research on floral development and we added that in discussion section. In this study, we would prefer to concentrate on traits related to plant size instead of flower count and floral development. 

6. Whether the crops were grown in greenhouse conditions after transgenic lines were made or in a growth chamber. All information should be provided.

We thank for your advice. We provided these information in updated version.

Minor comments

7. Please check carefully typos and minor errors throughout your MS as I can see at several places

Thank you. We made many corrections in this version.

8. Please mention full growth conditions since first time the seeds were germinated in greenhouse conditions.

We thank you for your comment. We revised this part in line 88-95.

9. Please mention what different time periods were selected for sampling

We thank you for your comment. We revised this part in line 110-116.

10. After the construction of constructs, how was genetic transformation done you have not mentioned in your methodology. The whole methodology should be presented. Besides, did you use co-infiltration method please mention.

Thank you for your suggestion. We provided reference related to tomato transformation in line 127. Besides, we transformed over-expression and knock-out construct separately to generate transgenic plants, instead of co-infiltration.

11. The results are not properly explained like how transgenic lines worked better compared to wild type.

Many thanks for your question. In order to better study the biological function of SlSPS in plant growth and response to the heat stress, SlSPS over-expression and knock-out plants were observed and there were several significant differences in terms of phenotypes and physiological indexes in comparison with wild-type.

12. I could not see any difference in H2O2 localization (DAB staining) preferably you can reanalyze it

We thank for your concern. Peroxidase in plant cells can react with hydrogen peroxide, which releases the oxygen to oxidize dimethyl-benzidine (DAB) to produce reddish brown sediment. The degree of DAB stain can significantly explain the accumulation of ROS in leaves. Using this principle, the leaves were used for detecting accumulation of hydrogen peroxide. SlSPS-overexpressing leaves showed lighter color than wild-type plants, while leaves of the knock-out plants exhibited deeper color than wild-type plants. These results indicated that SlSPS positively affects thermotolerance in tomato.

13. Correct the units of all bar diagrams like SOD, CAT, MDA etc.

Many thanks for your suggestion. We have checked the product manual and our protocol, and verified that the units were correct.

Reviewer 2 Report

The paper was about the characterization of tomato transgenic plants with overexpression or knockout mutations of the tomato SlSPS gene. The results showed interesting phenotypic changes in transgenic plants, suggesting that SlSPS can affect plant growth and heat tolerance.

Some comments to authors:

- Transgenic plants: Unfortunately, the author used the Micro-tom genotype to do transformation since this genotype is known to have many mutations that can affect plant growth. The author should try to transform to a more normal-looking genotype in the future. Also, should describe more; what is the T generation? Homozygosity? Number of plants...

- Phenotypic characterization: There is no mention of how replications were done for all of the data. Figure legends did not show statistics. Figure 3: Did the author record fruit yield? Again, it would be much better data if this was done with a normal genotype and actual field data.

Heat stress can also affect flowering development and fertilization process, such as pollen development. The author should try to do experiments on this too.

-Figure legends: Should expand more so the readers can understand independently from the text.

Author Response

Dear Reviewer,

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft. We have added necessary information to supplement our results and edited our article extensively. The detailed corrections are listed below.

1. Transgenic plants: Unfortunately, the author used the Micro-tom genotype to do transformation since this genotype is known to have many mutations that can affect plant growth. The author should try to transform to a more normal-looking genotype in the future. Also, should describe more; what is the T generation? Homozygosity? Number of plants...

We thank for your suggestion. Although Micro-Tom has many aspects of defects, its small size, convenience for genetic transformation and rapid reproduction make it a great model genotype on tomato research. Besides, we added related information in this version.

2.  Phenotypic characterization: There is no mention of how replications were done for all of the data. Figure legends did not show statistics. Figure 3: Did the author record fruit yield? Again, it would be much better data if this was done with a normal genotype and actual field data.

Heat stress can also affect flowering development and fertilization process, such as pollen development. The author should try to do experiments on this too.

We thank for your suggestion. We revised our manuscript and figure legend in this version. We will continue to study the mechanism of SlSPS in heat stress and the affection of the plant growth, including flowering development, fertilization process and fruit yield. We hope we can explain the function of SlSPS under heat stress in the future.

3. Figure legends: Should expand more so the readers can understand independently from the text.

We thank for your suggestion. We revised our figure legend in this version.

Reviewer 3 Report

In this study, authors investigated the role of a tomato sucrose phosphate synthase gene in the plant growth and heat stress tolerance. In my opinion, the study is planned nicely, and the findings are interesting. The data looks robust, and manuscript is written well. However, I have few comments that can be considered for improving the quality of manuscript.

·        I would suggest authors to make a separate paragraph explaining how heat stress treatment was given to the plants.

·         Please provide Solyc accession number of the characterized sucrose phosphate synthase gene.

·        I found several studies on the sucrose phosphate synthase genes. Few examples are here. https://www.sciencedirect.com/science/article/pii/S0981942899800230

·        https://academic.oup.com/plphys/article/113/4/1167/6070972 It will be good to include these studies in the discussion.

·        There are several typographical errors in the manuscript. Authors need to check them including the proper scientific notation carefully.

·        Please make a separate section for conclusion.

·        It would be good to include future prospects of the study in the conclusion section.

·        There are many places where the author's message is not clear due to improper phrasing and English language. Authors need to check that throughout the manuscript.

Author Response

Dear Reviewer,

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft. We have added necessary information to supplement our results and edited our article extensively. The detailed corrections are listed below.

1. I would suggest authors to make a separate paragraph explaining how heat stress treatment was given to the plants.

We thank for your suggestion. We revised “materials and methods” section in this version.

2. Please provide Solyc accession number of the characterized sucrose phosphate synthase gene.

We thank you for your comment. The accession number was given in “2.3.” and “3.1.”.

3. I found several studies on the sucrose phosphate synthase genes. Few examples are here. https://www.sciencedirect.com/science/article/pii/S0981942899800230

• https://academic.oup.com/plphys/article/113/4/1167/6070972 It will be good to include these studies in the discussion.

Thank you for your suggestion. We do believe these studies are valuable for our result. We included these studies in the discussion section.

4. There are several typographical errors in the manuscript. Authors need to check them including the proper scientific notation carefully.

We thank for your kind remind. We checked our manuscript and revised it.

5. Please make a separate section for conclusion.

We thank for your suggestion. We made a separate section for conclusion.

6. It would be good to include future prospects of the study in the conclusion section.

We thank for your comment. We supplement future prospects of the study in discussion part.

7. There are many places where the author's message is not clear due to improper phrasing and English language. Authors need to check that throughout the manuscript.

We thank for your comment and revised our manuscript.

Reviewer 4 Report

Dear Authors, thank you very much for the opportunity to take part in the review process of your paper. I think, that it is a very interesting paper, but further clarifications are necessary to make it understandable for wider audiences.

General comments:

I think that the title suggests, that SlSPS is the only gene, that regulates the investigated traits, which is obviously not true. I suggest modifying it, so that it will be clear for the reader, that it has influence besides others as well.

I suggest not using the phrase ’wild type’ in the case of the non-modified cultivar. It is very misleading as wild types are those plants which live in natural habitats without any direct human impact. A cultivated plant cannot be a wild type, especially a cultivar which was bred for laboratory experiments due to its small plant body size.

A general flaw of all figures, that abbreviations are not defined. Especially those of the treatments, OE and CR are undefined throughout the whole MS. Additionally, all the legends on the graphs are too small, which are unreadable in an A4 format -especially in the case of fig. 6, where CK and HS is undefined as well.

In figure 3, it would be useful, if the scale would be the same in the case of the seedling photos. Now it seems, that the control plants in the photos are not the same due to the different length.

The methodology of the statistical analysis is totally missing from the materials and methods.

It is not explained, why there are different numberings for the plants, are these repetitions or differ to some extent? I do not see the number of plants for sampling as well.

The discussion is a valuable contribution to the scientific progress on this topic; probably a short part could be added about the practical importance of the outcomes, including the higher BRIX, which is a determining parameter for industrial processing.

Author Response

Dear Reviewer,

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft. We have added necessary information to supplement our results and edited our article extensively. The detailed corrections are listed below.

1. I think that the title suggests, that SlSPS is the only gene, that regulates the investigated traits, which is obviously not true. I suggest modifying it, so that it will be clear for the reader, that it has influence besides others as well.

Many thanks for your suggestion.  We have changed the title “SlSPS, a sucrose phosphate synthase gene, mediates plant growth and thermotolerance in tomato”.

2. I suggest not using the phrase ’wild type’ in the case of the non-modified cultivar. It is very misleading as wild types are those plants which live in natural habitats without any direct human impact. A cultivated plant cannot be a wild type, especially a cultivar which was bred for laboratory experiments due to its small plant body size.

We thank for your suggestion and replaced “wild type” with “Micro-Tom”.

3. A general flaw of all figures, that abbreviations are not defined. Especially those of the treatments, OE and CR are undefined throughout the whole MS. Additionally, all the legends on the graphs are too small, which are unreadable in an A4 format -especially in the case of fig. 6, where CK and HS is undefined as well.

We thank for your suggestion. We revised the corresponding part in this version.

4. In figure 3, it would be useful, if the scale would be the same in the case of the seedling photos. Now it seems, that the control plants in the photos are not the same due to the different length.

We thank for your suggestion and modified the photos.

5. The methodology of the statistical analysis is totally missing from the materials and methods.

It is not explained, why there are different numberings for the plants, are these repetitions or differ to some extent? I do not see the number of plants for sampling as well.

We thank for your concern. We supplement the methodology of the statistical analysis in the caption of each figure. As in our study there was very primary statistical analysis, we prefer not to use a separate section to describe data analysis in materials and methods. Besides, different numberings for the plants represent the different lines of over-expression or knock-out transgenic plants, instead of repetitions. In the whole text, the plants with the same numbering represent the same line. For example, the results of SlSPS-CR1 in figure 2-3 are from the exact transgenic line in figure 1C. 

The discussion is a valuable contribution to the scientific progress on this topic; probably a short part could be added about the practical importance of the outcomes, including the higher BRIX, which is a determining parameter for industrial processing.

We thank for your suggestion and include the BRIX in the discussion section.

Round 2

Reviewer 1 Report

Authors have addressed all my comments and can be accepted

Reviewer 2 Report

The authors have spent effort and improved the manuscript