Overexpression of the SiLEA5 Gene in Saussurea involucrata Increases the Low-Temperature Tolerance of Transgenic Tomatoes

Round 1

Reviewer 1 Report

The manuscript “Overexpression of the SiLEA5 gene in Saussurea involucrata increases the low-temperature tolerance of transgenic tomatoes” represents an interesting research topic. This study by Liu et al. showed that SiLEA5 gene overexpression in tomato provides cold tolerance to tomato transgenic plants by increasing soluble sugar, soluble protein, and proline content and lowering MDA content. In transgenic tomato plants, CAT, SOD, and POD activity also increased significantly. Furthermore, authors have also demonstrated that in transgenic plants overexpression of SiLEA5 increased tomato yield by increasing the net photosynthetic rate. There is interesting data in the manuscript that could potentially be published after revision.

Specific comments:

1.      Introduce LEA gene family members (how many and their function briefly) and why the author chose LEA5 specifically.

2.      Line 39 LEA gene should be italicized

3.      Line 45-correct the language. mRNA or protein accumulation was already shown in the given reference.

4.      Line 60- Saussurea involucrate typographical error.

5.      Line 71-Laid instead of Laied. 

6.      Line 235- authors mentioned figure 1 as sequence analysis and domain conservation. However, Figure 1 is only showing phylogenetic analysis. Authors must show sequence analysis and domain conservation and why they named this gene as LEA5, either in Figure 1 or in supplementary data.

7.      Line 237- molecular weight of protein.

8.      There is no mention of Figure 1 in the result section.

9.      Line 252- what is qPCT and there is no figure for this data. Provide a proper heading for the result. This looks like the material and method section. In line 255- the authors mention “this further explained” however there is no data. Include RTPCR/qRTPCR to show the insertion and overexpression of the gene.

10.  Line 264-include recovery data in Figure 2.

11.  Line 299-Figure 2 Legend. The degree sign is wrong.

12.  In Figure legend 2B uses “8 h” while in 2C and D it’s “hours”. Maintain a consistent way of writing.

13.  Line 301 and 306- Mention biological or technical repeats.

14.  Line 308- Mention Figure No and give a proper heading to the result like other headings.

15.  Line 327-334- Explain the results according to Figure 4 (A-F).

16.  In all the figure legends, explain statistics as mentioned in the material and method section, especially in Figures 4 and 5.

17.  Line 346-2021 instead of 2020.

18.  Line 348-what is wild 2021?

19.  Line 381- In the discussion section, the authors wrote “SiLEA5 improved tolerance against cold, drought and salt stress” however, in the manuscript text there is no data for this conclusion.

20.  Line 413- In the discussion section, the authors mentioned that proline content is increased ten folds in transgenic plants. However, in the data, it doesn’t appear to be 10 folds. Provide the exact value.

21.  Line 432- Mention cold stress instead of abiotic stress. Abiotic stress is a broad term.

22.  Line 434-SiLEA5 should be italicized.  

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

In this work, the biological function of the protein SiLEA5 under abiotic stress (low temperature, drought and salt) was investigated, as well as its potential for use in agricultural breeding, using as model the medicinal plant Sassurea involucratas, which is resistant to cold temperatures. LEAs genes have been found to be upregulated at low temperatures in previous studies on Sassurea involucratas. LEA proteins have been also found to be abundant in plants under abiotic stress. To this end, tomato plants were transformed using SiLEA5 gene from Sassurea involucratas and subjected to low temperatures to investigate the biological pathways behind the role of this gene in low temperature response. The authors found evidence supporting the role of this gene in low temperature plant response by controlling proline metabolism and oxidative stress, as well as improving the photosynthetic capacity and productivity of transgenic tomato plants. I consider that this work fits the journal scope, provide an advancement of the current knowledge in plant stress resistance, and thus, the results will be of interest for the readers of this journal, so I recommend its publication. However, I have found several weaknesses (some of them very grave) that have to be fixed before its publication.

 

Abstract: I consider that the structure of the abstract should be reorganized. In order to facilitate the reading and understanding, it has to follow the same order as the main manuscript, by starting with an introduction, showing the context or background information for your research and the goals for your research; then the analytical methods used, your main findings (results) and finally, the significance or implications of your findings.

Line 16: “We simulated abiotic stress to determine the biological role of the SiLEA5 protein.”: What type of abiotic stress? Low temperatures? Please specify.

Line 18: “ At the same time, the two-year field statistical experiment…”: You mention the experiment as if it was already described for the reader, although it is the first time you talk about it. Please, provide a bit of context so that the reader can understand what this experiment is about.

Lines 23-24:  “it is abundantly obvious that our work establishes SiLEA5 protein as having significant scientific value and potential applications in crop breeding and research on stress resistance.”. I would recommend to modify the tone of this sentence by a more cautious one, e.g. “Thus, our work provides evidence for SiLEA5 protein to have a role in cold stress resistance in plants, as well as potential applications in crop breeding and research on cold stress resistance”. In addition, I would recommend to be more specific in all the manuscript when mentioning stress resistance by specifying that abiotic stress was tested and in particular, low temperature, salt and drought.

 

Introduction: Please provide more evidence/background to support your model plant species as a good model for this work

Lines 34-35: “More and more researchers use the means of plant genetic engineering to improve the stress resistance of crops to stabilize crop yield.”. The fact that many researchers use plant genetic engineering is not a valid argument to justify the use of genetic engineering to improve the stress resistance of crops (it is a fallacy, ad populum fallacy in particular). Please, provide a valid argument instead (for instance, that it has been proved in previous studies that genetic engineering speeds up the adaptation of crops to the new climatic conditions, also improving crop yields, and cite the referred bibliography).

 

Lines 45-46: “the mRNA of LEA gene will accumulate in a large amount (Wu et al., 2014; Zamora-Briseño and de Jiménez, 2016), which belongs to stress response proteins (Abdul Aziz et al., 2021).”. Please, explain more accurately this claim. The mRNA does not “belong” to a stress response protein but “translate” into a stress response protein.

 

Line 51: “LEA protein confers water resistance to mammalian somatic cells (Czernik et al., 51 2020).”. This is an article about plants, not animals. I recommend to remove this sentence

 

Lines 72-77: “In this study, we cloned SiLEA5 gene of Saussurea involucrata and obtained transgenic tomato lines by Agrobacterium-mediated  method. The low temperature stress test showed that the overexpression of SiLEA5 gene  improved the low temperature tolerance of tomato; the field test showed that the yield of tomato was increased. This research provides a scientific basis and extensive application value for the mining of stress resistance genes and the cultivation of new varieties.” This paragraph is a conclusion instead of an introduction. Please, provide the questions you want to answer (the hypotheses and main objectives of your work) instead of summarizing the results.

 

Material and Methods:

 

Lines 85-86: “MS solid medium and cultivated for 48h under dark conditions for tomato transformation. After the transformation of tomato…”. Please, explain a bit more how tomato transformation was performed (one line or two will be enough)

 

Lines 96-104: Please, provide the citations for all the software used. Use also a consistent verb tense in the paragraph (past simple, you wrote twice “is predicted” instead of “was predicted”)

 

Line 99: “NJ's (Neighbor-Joining)”. It should be stated the other way around (first the long name, then the short name)

 

Lines 154-156: “To evaluate the effects of abiotic stress on transgenic tomato plants, we selected wild-154 type and transgenic tomato plants with consistent growth at 4 weeks of age, which were treated with low temperature, drought, and salt, respectively.” I do not understand the term “respectively” here. I suppose that both controls and transgenic tomato plants were treated with low temperature, drought and salt treatments. Otherwise, the experimental design would not be correct (both controls and transgenic plants have to be subjected to the same treatments so that the controls are useful). If this is true, remove “respectively” here since it is not accurate. Otherwise, please, clarify what you mean.

 

Lines 154-170: You mentioned that low temperature, drought and salt treatments were performed. However, I do not see the drought nor salt treatments methods descriptions, only for low temperatures. Was drought implemented by freezing, I suppose? Please specify it so that it is clear for the reader. Same with the salt treatment. Use independent subheaders for each treatment description, called Low temperature treatment, Salt treatment and Drought treatment, so that it is clear for the reader. Avoid using colons in the main text (e.g. Lines 156-157: Low temperature treatment: wild-type and transgenic tomatoes grown at 25℃ _were transferred to 4◦C incubator 157 and treated with cold for 8 hours, treatment at 0℃ _for 6h.)

 

Lines 183-195: Please, use passive voice to describe the SOD activity assay. Avoid using colons.

 

Lines 211-212: Please, describe a bit what “normal field management” means (one sentence would be enough)

 

Lines 223-224: Please, state the specific number of plants measure. “At least 15 plants” is very vague.

 

Line 228: This statement about the p-values should be stated in the table showing the ANOVA results, not here.

 

Results: There is a major weakness here. You included discussion/conclusion statements in the results.

Lines 255-256: “We selected OE-2 and OE-3 lines for subsequent experiments.”. Why did you select these two lines in particular? You did not mention them in the Material and Methods nor in the results. Plus, I cannot access the supplementary material so I cannot check if these lines were previously mentioned there. Please, explain and if you could provide me access to the supplementary material, I would really appreciate it

 

Lines 265-268: “The ultimate degraded byproduct of membrane lipid peroxidation is malondialdehyde, and its concentration might indicate the severity of stress damage to plants.  Malondialdehyde and relative conductivity are crucial measures that assess the severity of bodily harm. Our findings demonstrated that MDA”. I can figure out that MDA means “Malondialdehyde” but please, state it together with the long word to avoid confusion (e.g. The ultimate degraded byproduct of membrane lipid peroxidation is malondialdehyde (MDA)…) and use the short form of the word (MDA) hereinafter.

 

Lines 288-290: “Plant antioxidant system is a major way to remove 288 toxic substances of reactive oxygen species in plants, which is significantly related to stress 289 resistance. We found that the CAT, SOD, and POD enzyme activities”. Again, please, state the short form (initials) of Reactive oxygen species (ROS, that you used afterwards) after the long form of the sentence to avoid confusion. Plus, the initials CAT, SOD and POD are not explained before. Please, use the long sentence before using initials.

 

Lines 295-296: “According to the aforementioned research, transgenic tomatoes 295 accumulate less ROS and are more resistant to cold temperatures.” This sentence is discussion, not results.

 

Lines 332-334: “These results indicated that SiLEA5 could improve photosynthesis rate and water use efficiency by regulating intercellular CO2 concentration and stomatal conductance.” Again, this sentence has to be placed in discussion not results

 

Lines 357-364: “In conclusion, the SiLEA5 gene increased tomato yield. This is probably because the SiLEA5 gene directly encouraged cell division and growth, which in turn increased the number of fruits per plant. Additionally, the increased transverse and longitudinal diameter length of the transgenic tomatoes also increased the weight of the tomato fruit (Fig. 5F-H). Not only is tomato output correlated with farmers' yearly incomes, but it is also an important breeding indicator. As a result of our research, it is possible to use the SiLEA5 gene as a gene resource for creating new high-yielding, stress-tolerant crop types.” Again, these sentences are discussion/conclusions, not results. Please, place all of the in the discussion/conclusion sections. In addition, I would rephrase the last sentence by using a more moderate tone and accurate statements (e.g. Our results suggest that SiLEA5 gene can be effectively used to develop high-yielding, stress-tolerant transgenic crop varieties).

 

Discussion: There are incongruences in your discussion with your results (and even your title!). You claimed that your results support that the transgenic plants showed more tolerance to low temperatures, drought and salt stresses, whereas your results and title only support/claims an enhanced tolerance to low temperatures. Please, clarify or fix accordingly. Some references are also lacking to support your claims/conclusions.

Lines 376-377: “The Saussurea involucrata created and selected a physiological mechanism that could withstand the harsh environment (Gong et al., 2019).” This sentence would be more appropriate for the introduction, in order to explain why this plant species is a good model for this study. You can explain this mechanism in more detail in the introduction and leave this short sentence as a reminder in the discussion.

 

Lines 379-380: “We discovered that transgenic plants overexpressing SiLEA5 displayed some tolerance under low temperature, drought, and moderate salt stress.” According to your results (and even title!)  SiLEA5 gene led to higher tolerance to low temperature in transgenic plants, not drought nor moderate salt stress (even if both abiotic stresses were tested in the experiments and analyzed). Please, remove this statement or explain/clarify in the results the results supporting these claims.

 

Lines 388-389: “The introduction of the stress-resistant gene may essentially resolve this issue and enhance the plant's capacity to withstand stress.” What stress-resistant gene, SiLEA5? Please, clarify it to avoid confusion.

 

Lines 400-402: “This study discovered that the accumulation of MDA in transgenic tomatoes was 400 significantly lower than that in wild-type tomatoes under conditions of increased low 401 temperature, drought, and salt stress.” Again, your results only suggest that this happen under low temperature. Please, clarify it.

 

Line 405: What ZmSAMDC gene has to do with LEA proteins? Please, explain.

 

Lines 444-448: “Therefore, we propose that SiLEA5 protein may participate in ABA signaling through the expression regulation of the CBF/DREB1 transcription factor family and fur ther participate in the response to stress signals (Ali and Hadi, 2018; Cao et al., 2020; Hassan et al., 2021), it is of great scientific significance to better understand the physiological response mechanism of plants to stress environment.” Do you have any evidence to support this claim? I know that the photosynthetic rate and the gas exchange was higher in transgenic plants, but ABA is not the only phytohormone implied in the stomatal opening regulation. Please, reformulate this claim or provide evidence for it.

 

Line 449: “At present, the light energy utilization efficiency of field crops is still very low, far from reaching the theoretical value.” A citation for this claim is lacking. Please provide the source.

 

Line 476: “Traditional methods of crop improvement may have reached its limits”. Please, provide evidence/citations to support this claim.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors declares the overexpression of the SiLEA5 gene in Saussurea involucrata increases the low temperature tolerance of transgenic tomatoes.

I have few comments related to the manuscipt:

*Please look at the highlighted paragraphs...there are also similar technical and grammatical errors throughout the manuscript.

*Please try to avoid long sentences...better to make simple and clear...

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Dear authors,

Thank you very much for your thoughtful revision of the article. I consider that the quality of the manuscript has substantially improved after your last revision, and now it fits the journal scope and scientific quality requirements. I also consider that it provides an advancement of the current knowledge in plant stress resistance, and thus, the results will be of interest for the readers of this journal. Thus, I recommend its publication in its present form.