Identification of bHLH Family Genes in Rhododendron hainanense Merr. and Verification of Their Role in Heat Tolerance
, Minghui Zhai, Yufei Li, Yifan Li, Jiaxuan Shi, Xueping Feng, Jian Wang, Xiqiang Song, Youhai Shi and Ying Zhao *
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe work Verification of the role of bHLH family genes in heat tolerance of Rhododendron hainanense Merr. through transcriptome and metabolome analysis provides relevant information about the species and its relationship with the bHLH gene family. The article has the necessary experiments to support the research findings; however, it is relevant to make some corrections that improve the writing quality. Generally, the authors must follow the authors' instructions for the references. On the other hand, they need to review the statistical analysis and its graphs; this suggestion is vital to resolve it because the results must be substantiated with the smallest margin of error. The rest of the recommendations are listed as follows:
117, 118: Check the correct name of the species in V2 is R. dauricum, and in V1 it is another name
124: correct R. Hainamense; the correct one is R. hainamense
218: There are two points, remove one (The data acquasition …)
238: put the sign of the ions in superscript
261: Put the R in the capital letter of the word “Results.”
Figure 4: It is not legible
512: Put in bold “Figure 12”
529: put in bold “Figure 13”
531: put in bold “Figure 14”
538, 539: put in italics and lowercase the P of probability or p-value
Table 3: put in italics the letter O and the p in the names of the compounds
Figure 15: it is not legible, it is immensely loaded with information, and it is not appreciated
Figure 16: not legible
567: Write in bold “Figure 16”
Figure 17: review the data of the relative expression of bHLH89, the standard deviations or error bars on days 3 and 7 are excessively high, and on day 5 it strangely decreases. Or improve the description of the result to explain this statistically atypical behavior. On the other hand, this figure could be sectioned into a), b), c), d), etc.
References: they do not have the format of the journal; please review the instructions for the authors
Comments on the Quality of English LanguageThe syntax of the document is sufficient to understand the content.
Author Response
Comment1: 117, 118: Check the correct name of the species in V2 is R. dauricum, and in V1 it is another name
Response1: In V2, R. dauricum, V1 is Rhododendron chinensis, and Rhododendron sinensis is a close relative of Rhododendron chinensis, and we compare the two to evaluate heat tolerance.
Comment2: 124: correct R. Hainamense; the correct one is R. hainamense
Response2: Agreed, the error has been corrected.
Comment3: 218: There are two points, remove one (The data acquasition …)
Response3:Agree.the error has been corrected.
Comment4: 238: put the sign of the ions in superscript
Response4:Agree.the error has been corrected.
Comment5:261: Put the R in the capital letter of the word “Results.”
Response5:Agree.the error has been corrected.
Comment6:Figure 4: It is not legible
Response6: We have updated the picture illustration of the position of the bHLH gene on the chromosome, and now it is clearer.
Comment7:512: Put in bold “Figure 12”
Response7:Agree.the error has been corrected.
Comment8:529: put in bold “Figure 13”
Response8:Agree.the error has been corrected.
Comment9:531: put in bold “Figure 14”
Response9:Agree.the error has been corrected.
Comment10:538, 539: put in italics and lowercase the P of probability or p-value
Response10:Agree.the error has been corrected.
Comment11:Table 3: put in italics the letter O and the p in the names of the compounds
Response11:Agree.the error has been corrected.
Comment12:Figure 15: it is not legible, it is immensely loaded with information, and it is not appreciated
Response12: Agree, we have generated a clearer image that shows the correlation between key bHLH genes and differential metabolites during heat treatment.
Comment13:Figure 16: not legible
Response13:Agree.the error has been corrected.
Comment14:567: Write in bold “Figure 16”
Response14:Agree.the error has been corrected.
Comment15:Figure 17: review the data of the relative expression of bHLH89, the standard deviations or error bars on days 3 and 7 are excessively high, and on day 5 it strangely decreases. Or improve the description of the result to explain this statistically atypical behavior. On the other hand, this figure could be sectioned into a), b), c), d), etc.
Response15：Agree, if it's an experimental error, then we still need to repeat the experiment to verify that this atypical change will occur.
Comment16：References: they do not have the format of the journal; please review the instructions for the authors
Response16：Agree, the error has been corrected.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript "Verification of the role of bHLH family genes in heat tolerance of Rhododendron hainanense Merr. through transcriptome and metabolome analysis." is devoted to the study of the bHLH transcription factor gene family and to determine the extent of their participation in heat stress resistance in Rhododendron hainanense. The topic is interesting, a huge amount of work has been done, the transcriptome and metabolome of plants have been obtained and analyzed, candidate genes participating in the response to heat stress and key metabolites have been identified. The work may be of interest to a wide range of readers, but there are many comments on the manuscript.
1. A significant part of the results is devoted to the study of the gene family itself, and not only to the verification of their role in resistance to high temperatures, therefore the title of the article does not fully reflect its content.
2. Line 60, 61 – missing spaces in references
3. Lines 61 – 68 It is not very clear how the groups and families of bHLH genes are related. Perhaps, to clarify this point, it is necessary to indicate in whom Ledent identified and divided the bHLH genes into groups – in plants or in animals.
4. Line 88 – Part of the reference is missing.
5. Line 115 -80℃ Celsius – the letter C already means degrees Celsius, it is not necessary to write Celsius a second time.
6. Although R. dauricum was used as a control for comparison, its source should also be indicated in the Material and Methods section. I would also like to say a few words about why this particular species was chosen for comparison.
7. In the description of the plant material and sampling, it is necessary to specify how much material was taken for analysis? Why is Figure 1 provided if it is not mentioned or discussed anywhere in the text of the article? If we are discussing the phenotype of plants in the photo, can the phenotype visible in the photo be partly explained by the fact that by the end of the experiment, the leaves of the plants had already been taken for analysis for the 9th time in a row?
8. Bring the species name to uniformity - the text contains both R. Hainanense (the capital H is superfluous) and R.hainanense (there is no space after the period). Moreover, later in the discussion the name Hainan azalea also appears, and nowhere is it clearly indicated whether this is the name of the same species, i.e. R. hainanense.
9. Line 128 – thaliana is written twice.
10. Lines 138-139 the sentence appears unfinished or incoherent.
11. Lines 141-155 – Tbtools – to bring to a single spelling, indicate the program version in brackets right after its mention. These lines are massively missing spaces. BLAST is an abbreviation, all letters are capitalized. The previous subsections in Materials and Methods are written in the past tense, this one in the present, which also looks strange.
12. Section 2.1 states that samples were collected at 9 points from day 0 to day 7 of the experiment. Section 2.6 states that RNA was isolated from leaves collected on days 0 and 7. It is not immediately clear why samples were collected at other points in the experiment and whether RNA was isolated from all collected samples. All these questions are clarified only at the end of the section. Perhaps it is worth adding a drawing with the experiment scheme, how many plants, at what age of plants the experiment began, how many control points in the experiment, at what stages samples were taken and for what analysis.
13. Section 2.7 does not indicate which samples were taken from which plants, or by what principle they were divided into 2 groups. Were these samples taken during the temperature exposure experiment? What amount of material was taken from which part of the plants?
14. Although, judging by the notations on the graphs, statistical processing of the data was carried out, there is no mention anywhere of what methods and programs were used for statistical analysis.
15. The first paragraph of subsection 3.1 is more suitable for the Material and Methods section, where a description of the plant species used in the work would be appropriate.
16. The Materials and Methods section does not describe how the analysis of SOD activity, MDA content, hydrogen peroxide content and proline content was performed. There is no decoding of SOD and MDA at the first mention in the text. The diagrams in Figure 2 should be designated with letters and accordingly referred to in the text - Figure 2A, etc.
17. Lines 333-334 – the sentence needs to be rewritten. In its current form, it simply encourages readers to use Expasy online for analysis. In addition, in its current form, the sentence is more consistent with the Materials and Methods section, which already states that Expasy online was used to evaluate the physicochemical properties of proteins.
18. Line 339 - in vitro – Latin expressions should be written in italics.
19. There is no reference to Figure 3 in the text.
20. Line 353 - is there a typo here? Have you annotated the gff file of Hainan R. dauricum and looked at the chromosome distribution of 117 genes of R. hainanense?
21. The Discussion section is very short, although even the Results section contains suggestions that could be developed more widely in the discussion. For example: This suggests that the chromosomes with the majority of RhbHLH genes are more likely to be related to plant growth and stress processes…. It is noteworthy that all the kaks values ​​of the duplicated segments were less than 1, indicating that they have evolved under purifying selection. This may be one of the driving forces of evolution…. Cis-regulatory elements in organisms regulate gene expression by providing binding sites for interaction with other proteins. Therefore, different binding sites interact with proteins of varying functions, determining the function of the cis-regulatory element...
22. The text must contain references to all figures! First of all, this concerns the figures in the Results section, most of them are not mentioned in the text, which raises the question of their necessity for the article.
23. Lines 385-388 – missing italics in the Latin names of species.
24. Figures 8 and 9 are very small, although they contain a lot of information. In this form, it is quite difficult to evaluate them. Especially figure 9, where the colors designating individual elements are almost indistinguishable.
25. Lines 433-434 – the sentence needs to be reformulated, as it is not very clear in its current form.
26. Line 441 – the first time the abbreviation (CRE) is mentioned, it must be deciphered.
27. Lines 4446 and 451 - the same abbreviation (LTR) is deciphered differently.
28. Lines 443 and 453 – G-box and g – box spelling must be unified.
29. It is not very clear why a visualization of the protein structure is provided if no further conclusions are drawn from these results and the data in the work are discussed little, except for general conclusions such as - proteins from different subfamilies differ in structure.
30. Lines 472-485 seem more appropriate for the Discussion section, since the obtained data are compared (discussed) with information obtained in other works and experiments. In this section, authors should limit themselves to a description of the network they have created: which genes are key, which have more/fewer connections, and is it possible to somehow link these data with other research results?
31. Line 491 – PebHLH – is this a typo?
32. Lines 493-494 – in the Material and Methods section, where the transcriptome analysis is described, it is necessary to somehow decipher the designations of the samples RhTH1, RhTH2, RhTH3 and RhTN1, RhTN2, RhTN3 – otherwise it is not clear what is hidden behind these designations and, accordingly, it is difficult to understand the results obtained.
33. The figures following one after another, without text between them, should be combined into one figure of two parts. For example, figures 13 and 14. In addition, and all designations (for example, designations of groups RhMN RhMH) should be deciphered in the figure captions.
34. It would be logical for section 3.9 to follow section 3.6 describing transcriptomic analysis. As it stands, the narrative seems disjointed – it talks about the transcriptome, then the metabolome, and then returns to expression.
35. Section 3.9 Real-time Fluorescence Quantitative Verification describes not only verification but also changes in the expression level of a selected group of genes during stress. Perhaps the title should be changed.
36. The sentence "It is well known that flavonoids are crucial in plant temperature stress." requires a reference.
37. Line 601 is the first time Hainan azalea is mentioned in the article; readers who are not so familiar with these plants may not understand what is being discussed without the Latin name of the species.
38. The Discussion section is very short and contains very few references (only 8 sources). Basically, a small part of the work concerning transcriptomic and metabolomic analysis is discussed. A significant part of the results is not discussed at all. In particular, sections concerning gene localization on chromosomes, gene duplication, the constructed gene network, 3D protein structures, cis-motifs are not discussed. The question arises - if the data are not discussed at all, are they necessary in this work?
Author Response
Comment 1： A significant part of the results is devoted to the study of the gene family itself, and not only to the verification of their role in resistance to high temperatures, therefore the title of the article does not fully reflect its content.
Response 1：Agree, we have revised the article title and added bHLH family identification.[Line2-3]
Comment 2：Line 60, 61 – missing spaces in references.
Response 2：Agree, the error has been corrected.[Line 62-63]
Comment 3： Lines 61 – 68 It is not very clear how the groups and families of bHLH genes are related. Perhaps, to clarify this point, it is necessary to indicate in whom Ledent identified and divided the bHLH genes into groups – in plants or in animals.
Response 3：Agree. Li T divides all bHLH genes in rice and Arabidopsis into 25 families.[Line 69-70]
Comment 4：Line 88 – Part of the reference is missing.
Response 4：Agree, the error has been corrected.[Line 90]
Comment 5： Line 115-80℃ Celsius – the letter C already means degrees Celsius, it is not necessary to write Celsius a second time.
Response 5：Agree, the error has been corrected.[line 117]
Comment 6： Although R. dauricum was used as a control for comparison, its source should also be indicated in the Material and Methods section. I would also like to say a few words about why this particular species was chosen for comparison.
Response 6：Agree. R. Dauricum also comes from the Agricultural Science Base of Hainan University and was purchased together with Hainan Rhododendron from Jinhua Yonggen Rhododendron Company in 2020. The reason for choosing this species is mentioned in section 3.1，R. dauricum is a plant in the Ericaceae family, and it is mentioned in the literature that R. dauricum and R. hainanense belong to the same genus, showing a close evolutionary relationship. It is currently widely used in landscape gardening. Comparisons between Rhododendron species reveal that R. dauricum has excellent heat tolerance , making it a suitable control material for studying the heat tolerance of R. hainanense.[ Line 290]
Comment 7： In the description of the plant material and sampling, it is necessary to specify how much material was taken for analysis? Why is Figure 1 provided if it is not mentioned or discussed anywhere in the text of the article? If we are discussing the phenotype of plants in the photo, can the phenotype visible in the photo be partly explained by the fact that by the end of the experiment, the leaves of the plants had already been taken for analysis for the 9th time in a row?
Response 7：agree. We have removed the description of the quantity of materials. Figure 1 aims to express the phenotypic changes of Hainan Rhododendron under high temperature treatment. We have described Figure 1 The leaves in the picture were indeed photographed 9 times.
Comment 8：Bring the species name to uniformity - the text contains both R. Hainanense (the capital H is superfluous) and R.hainanense (there is no space after the period). Moreover, later in the discussion the name Hainan azalea also appears, and nowhere is it clearly indicated whether this is the name of the same species, i.e. R. hainanense.
Response 8：Agree, we have made modifications to R Hainanese (uppercase H) and space issues. Hainan Rhododendron was our spelling mistake, which has been corrected to Hainan Rhododendron.
Comment9: Line 128 – thaliana is written twice.
Response9: Agree, the error has been corrected.
Comment10 :Lines 138-139 the sentence appears unfinished or incoherent.
Response10:Agree, the error has been corrected.
Comment11: Lines 141-155 – Tbtools – to bring to a single spelling, indicate the program version in brackets right after its mention. These lines are massively missing spaces. BLAST is an abbreviation, all letters are capitalized. The previous subsections in Materials and Methods are written in the past tense, this one in the present, which also looks strange.
Response11 :Agree, changed the position of the version number, added spaces to the appropriate position, and changed it to BLASTp. The tense of this section has been changed to the past tense.
Comment 12：Section 2.1 states that samples were collected at 9 points from day 0 to day 7 of the experiment. Section 2.6 states that RNA was isolated from leaves collected on days 0 and 7. It is not immediately clear why samples were collected at other points in the experiment and whether RNA was isolated from all collected samples. All these questions are clarified only at the end of the section. Perhaps it is worth adding a drawing with the experiment scheme, how many plants, at what age of plants the experiment began, how many control points in the experiment, at what stages samples were taken and for what analysis.
Response 12：We collected samples from day 0 to day 7 and isolated RNA from all samples (9 time points). In Section 2.6, only materials from Day 0 and Day 7 were selected for transcriptome sequencing to search for differentially expressed genes and metabolites over a certain time span.
Comment 13：Section 2.7 does not indicate which samples were taken from which plants, or by what principle they were divided into 2 groups. Were these samples taken during the temperature exposure experiment? What amount of material was taken from which part of the plants?
Response 13：agree. The samples were obtained from the leaf tissue of Rhododendron hainanense, divided into RHTN and RHTH, representing tissues treated at normal temperature and high temperature.
Comment 14：Although, judging by the notations on the graphs, statistical processing of the data was carried out, there is no mention anywhere of what methods and programs were used for statistical analysis.
Response 14：Agreed, we used SPSS for statistical analysis.
Comment 15：The first paragraph of subsection 3.1 is more suitable for the Material and Methods section, where a description of the plant species used in the work would be appropriate.
Response 15：Agreed, we will place it in the Materials and Methods section.
Comment 16：The Materials and Methods section does not describe how the analysis of SOD activity, MDA content, hydrogen peroxide content and proline content was performed. There is no decoding of SOD and MDA at the first mention in the text. The diagrams in Figure 2 should be designated with letters and accordingly referred to in the text - Figure 2A, etc.
Response 16：agree. Methods for analyzing physiological indicators have been added after Chapter 2.1. And modify the subsequent formatting issues.
Comment 17：Lines 333-334 – the sentence needs to be rewritten. In its current form, it simply encourages readers to use Expasy online for analysis. In addition, in its current form, the sentence is more consistent with the Materials and Methods section, which already states that Expasy online was used to evaluate the physicochemical properties of proteins.
Response 17：Agree. Figure 3 shows the amino acid conservation of the bHLH conserved sequence in Hainan Rhododendron and its standard protein conformation, which we have added to the second section.
Comment 18：Line 339 - in vitro – Latin expressions should be written in italics.
Response 18：Agree, the error has been corrected.
Comment 19：There is no reference to Figure 3 in the text.
Response 19：Agree, the error has been corrected.
Comment 20：Line 353 - is there a typo here? Have you annotated the gff file of Hainan R. dauricum and looked at the chromosome distribution of 117 genes of R. hainanense?
Response 20：Agree, the error has been corrected.This is a spelling error, we have annotated R. hainanense gff file.
Comment 21： The Discussion section is very short, although even the Results section contains suggestions that could be developed more widely in the discussion. For example: This suggests that the chromosomes with the majority of RhbHLH genes are more likely to be related to plant growth and stress processes…. It is noteworthy that all the kaks values ​​of the duplicated segments were less than 1, indicating that they have evolved under purifying selection. This may be one of the driving forces of evolution…. Cis-regulatory elements in organisms regulate gene expression by providing binding sites for interaction with other proteins. Therefore, different binding sites interact with proteins of varying functions, determining the function of the cis-regulatory element...
Response 21：Agree. We have expanded the content of the discussion section, allowing him to discuss more other parts of the article.
Comment 22：The text must contain references to all figures! First of all, this concerns the figures in the Results section, most of them are not mentioned in the text, which raises the question of their necessity for the article.
Response 22：Agree, the error has been corrected.
Comment 23：Lines 385-388 – missing italics in the Latin names of species.
Response 23 ：Agree, the error has been corrected.
Comment 24： Figures 8 and 9 are very small, although they contain a lot of information. In this form, it is quite difficult to evaluate them. Especially figure 9, where the colors designating individual elements are almost indistinguishable.
Response 24：Agree, We have regenerated clearer images to make them easier to understand.
Comment 25： Lines 433-434 – the sentence needs to be reformulated, as it is not very clear in its current form.
Response 25：Agree, the error has been corrected.
Comment26：Line 441 – the first time the abbreviation (CRE) is mentioned, it must be deciphered.
Response26：Agree, the error has been corrected.
Comment27： Lines 4446 and 451 - the same abbreviation (LTR) is deciphered differently.
Response27：Agree, the error has been corrected.
Comment28： Lines 443 and 453 – G-box and g – box spelling must be unified.
Response28：Agree, the error has been corrected.
Comment29：It is not very clear why a visualization of the protein structure is provided if no further conclusions are drawn from these results and the data in the work are discussed little, except for general conclusions such as - proteins from different subfamilies differ in structure.
Response29：Agree, the error has been corrected.
Comment30：Lines 472-485 seem more appropriate for the Discussion section, since the obtained data are compared (discussed) with information obtained in other works and experiments. In this section, authors should limit themselves to a description of the network they have created: which genes are key, which have more/fewer connections, and is it possible to somehow link these data with other research results?
Response30：Agree, the error has been corrected.
Comment31：Line 491 – PebHLH – is this a typo?
Response31：Agree, This is a spelling error, the error has been corrected.
Comment32：Lines 493-494 – in the Material and Methods section, where the transcriptome analysis is described, it is necessary to somehow decipher the designations of the samples RhTH1, RhTH2, RhTH3 and RhTN1, RhTN2, RhTN3 – otherwise it is not clear what is hidden behind these designations and, accordingly, it is difficult to understand the results obtained.
Response32：Agree,In section 2.7, we provided comments on it.
Comment33：The figures following one after another, without text between them, should be combined into one figure of two parts. For example, figures 13 and 14. In addition, and all designations (for example, designations of groups RhMN RhMH) should be deciphered in the figure captions.
Response33: Agree, the error has been corrected.
Comment34：It would be logical for section 3.9 to follow section 3.6 describing transcriptomic analysis. As it stands, the narrative seems disjointed – it talks about the transcriptome, then the metabolome, and then returns to expression.
Response34：Agree, we adjusted the before and after logic to make it seem more reasonable.
Comment35：Section 3.9 Real-time Fluorescence Quantitative Verification describes not only verification but also changes in the expression level of a selected group of genes during stress. Perhaps the title should be changed.
Response35：Agree, we changed the title to increase the relative expression level of the bHLH gene.
Commnet36:The sentence "It is well known that flavonoids are crucial in plant temperature stress." requires a reference.
Response36: Agree, the correct references have been added.
Comment37: Line 601 is the first time Hainan azalea is mentioned in the article; readers who are not so familiar with these plants may not understand what is being discussed without the Latin name of the species.
Response37: Agree, line 601 is also R. hainanense .
Comment38： The Discussion section is very short and contains very few references (only 8 sources). Basically, a small part of the work concerning transcriptomic and metabolomic analysis is discussed. A significant part of the results is not discussed at all. In particular, sections concerning gene localization on chromosomes, gene duplication, the constructed gene network, 3D protein structures, cis-motifs are not discussed. The question arises - if the data are not discussed at all, are they necessary in this work?
Response38： Agreed, thanks for the suggestion. We've added a discussion section to discuss motif composition, collinearity events, and protein-protein interaction networks. （line617-639）
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article is devoted to the analysis of the bHLH trans-factor gene family in the genome of Rhododendron hainanense Merr under conditions of elevated temperature. These trans-factors are involved in the regulation of plant growth and development, as well as in plant stress resistance. 117 genes have been identified and a complete bioinformatic characterisation of both the genes and the proteins they encode has been given. Some physiological characteristics of plant response to elevated temperature are given. Transcriptomic and metabolomic studies were carried out. The relationship between the expression of certain genes and metabolites related to these genes has been established. Overall, the work is very large and useful. After some editing, the article can be accepted for publication.
I would like to draw the attention of the authors to the following shortcomings.
1. The “Methods” part does not describe the determination of H2O2, proline, MDA and SOD activity, which is absolutely necessary.
2. In section 2.8, give at least some basic indicators of qRT-PCR performance. For example, reference genes, etc.
3. It would be good to brighten up Fig. 1. There is a tenth pot of plants in each row. What is this variant? Why is the 10th pot almost always the larger pot?
4. Fig. 2. The captions in green are poorly readable. It is necessary to make correct captions of the ordinate axes. For example: Proline content and then the units of measurement. On the abscissa axes the duration of heat treatment is indicated, so it should be signed like that.
5. Why not place the fragments of Figure 2 in the order they are discussed in the text or change the sequence of discussion as in Figure 2?
6. The MDA content after 3 days at Rd in Fig. 2 may well be random, given the large deviations and only one point with elevated values. If we accept this possibility, the interpretation of the results changes. The increase in SOD activity in Rh after 7 days in the same figure against a background of stable levels is questionable.
7. In Fig. 4 it is better to sign the chromosome numbers
8. I don't know if it is possible to correct Fig. 8. It is impossible to read this way, when enlarged up to 400% the sharpness disappears and it is impossible to read again
9. Line 297 - (Figure) indicate figure number
10. Line 110 - 2000 lx would be good to convert to μmol m-2 s-1
Author Response
Comment 1：The “Methods” part does not describe the determination of H2O2, proline, MDA and SOD activity, which is absolutely necessary.
Response 1：Agree, methods for measuring H2O2, proline, MDA, and SOD activity have been added after section 2.1.
Comment 2：In section 2.8, give at least some basic indicators of qRT-PCR performance. For example, reference genes, etc.
Response 2：Agree, At the end of section 2.8, it is indicated that 18s is used as the housekeeping gene.
Comment 3： It would be good to brighten up Fig. 1. There is a tenth pot of plants in each row. What is this variant? Why is the 10th pot almost always the larger pot?
Response 3：These ten pots of plants are not much different, except that the tenth pot is larger in size.
Comment 4： Fig. 2. The captions in green are poorly readable. It is necessary to make correct captions of the ordinate axes. For example: Proline content and then the units of measurement. On the abscissa axes the duration of heat treatment is indicated, so it should be signed like that.
Response 4：Agree, the image has been modified
Comment 5：Why not place the fragments of Figure 2 in the order they are discussed in the text or change the sequence of discussion as in Figure 2?
Response 5：Agree, the display order in the image has been corrected
Comment 6：The MDA content after 3 days at Rd in Fig. 2 may well be random, given the large deviations and only one point with elevated values. If we accept this possibility, the interpretation of the results changes. The increase in SOD activity in Rh after 7 days in the same figure against a background of stable levels is questionable.
Response 6：Agree. if it is an experimental error, then we still need to conduct repeated experiments to verify whether such atypical changes will occur.
Comment 7：In Fig. 4 it is better to sign the chromosome numbers
Response 7：Agree, the image has been modified.
Comment 8： I don't know if it is possible to correct Fig. 8. It is impossible to read this way, when enlarged up to 400% the sharpness disappears and it is impossible to read again.
Response 8：Agree, we have regenerated the image with higher clarity.
Comment 9：Line 297 - (Figure) indicate figure number
Response 9：Agree, revised to Figure 2.
Comment 10： Line 110 - 2000 lx would be good to convert to μmol m-2 s-1.
Response 10：Agree, it has been changed to μmol m-2 s-1.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article has certainly improved after the edits were made. The narrative has become more logical and consistent and makes a more coherent impression. However, there are still a few comments left:
1. The Latin name of the species must be written in italics, including in the title.
2. The name of the biotechnological kit must be added to line 132.
3. Unify the spelling of TBtools or Tbtools. The same with the spelling of MCScanX.
4. Lines 162-163 are the Latin names, they must be written in italics.
5. The first mention of the Latin name of rice was in line 72, where it should be mentioned in full, then it should be abbreviated (for example, in line 163, the full name should no longer be used).
6. Lines 273-275 should also be in the past tense.
7. Something is apparently missing in the sentence on lines 348-350, perhaps around the word that on line 348. The same thing in line 352 - the sentence needs to be edited.
8. It would be good to add a reference to literature to the sentence on lines 588-589
Author Response
Comment1:The Latin name of the species must be written in italics, including in the title.
Response1: Agree, we have modified the Latin name in the title to italic format.(line2)
Comment2:The name of the biotechnological kit must be added to line 132.
Response2:Agreed, we have added the name and source of the reagent kit at the end of the paragraph.(line137-140)
Comment3:Unify the spelling of TBtools or Tbtools. The same with the spelling of MCScanX.
Response3:Agreed, we have unified the names TBtools and MCScanX throughout the entire document.
Comment4:Lines 162-163 are the Latin names, they must be written in italics.
Response4:Agreed, the format has been modified.(line172-179)
Comment5:The first mention of the Latin name of rice was in line 72, where it should be mentioned in full, then it should be abbreviated (for example, in line 163, the full name should no longer be used).
Response5:Agreed, we have corrected the Latin name format error.(line73-74)
Comment6:Lines 273-275 should also be in the past tense.
Response6:Agreed, we will modify the tense of this paragraph to the past tense.(line275-284)
Comment7:Something is apparently missing in the sentence on lines 348-350, perhaps around the word that on line 348. The same thing in line 352 - the sentence needs to be edited.
Response7:Agreed, we have improved this section.(line353-357)
Comment8:It would be good to add a reference to literature to the sentence on lines 588-589
Response8:Agreed, we have added two references in that area.(line606-608)
