The Copper Chaperone Protein Gene GmATX1 Promotes Seed Vigor and Seedling Tolerance under Heavy Metal and High Temperature and Humidity Stresses in Transgenic Arabidopsis

Round 1

Reviewer 1 Report

Please kindly find the comments for the authors in the attachment.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer:

 

Thank you for your valuable and thoughtful comments and suggestions for our manuscript. We appreciate your excellent comments and suggestions. We have considered all of these suggestions and comments in revising our manuscript. The following lists our responses to your comments.

 

Best Wishes,

 

Hao Ma

 

State Key Lab of Crop Genetics and Germplasm Enhancement,

Nanjing Agricultural University,

Nanjing, Jiangsu Province 210095, China

 

 

Answer to the reviewers’ comments:

 

1. In our a previous study, by differential proteomic analysis of leaves, cotyledons and embryos during seed development of soybean cultivars Ningzhen No. 1 and Xiangdou No. 3 under high temperature and humidity (HTH) stress, respectively, a key copper chaperone protein, GmATX1, was identified in seed – lack of the reference, lines 69-72

Answer: the reference has been added.

 

Results.

2. Seeds were collected from the central part of 10 control plants and 10 treated plants after treated for 24, 96, and 168 h, respectively, and each sample contains three independent biological replicates – lines 374-376, lack of the explanations, why in these time points.

Answer: The time points were selected according to our previous study, and at these time points, the developing seed of soybean was found to respond strongly to HTH stress in morphologic, physiological and biochemical, and molecular levels [7].

 

3. The results showed that compared to these of the controls, the expression levels of GmATX1 were very significantly (p < 0.01) increased at all the stress time points in the seeds of Xiangdou No. 3 and only at the stress time points of 96 and 168 h in the seeds of Ningzhen No. 1 (Figure 2AB). Lines 103-106, please explain why in one line – at all the stress time points, in the second – only at two time points.

Answer: this may be due to there were differences in genetic background between varieties. We have added a paragraph to discuss this in Discussion.

 

4. Total RNAs from treatments and controls containing four biological replicates were extracted using the Universal Plant RNA Extraction Kit (Vazyme Biotech, China) according to the manufacturer's instructions. Lines 321-323, How RNA was isolated from seeds? Any differences from leaf RNA isolation?

Answer: How RNA was isolated from seed has been added in Material and Methods.

 

5. The highest expression level of GmATX1 was found in pods, followed by in mature seeds and flowers in Xiangdou No. 3 (Figure 2C), while in seeds, followed by in young pods and flowers in Ningzhen No. 1 (Figure 2D). Lines 117-119, Please specify what does mean young seed?, mature seeds?

Answer: young seed, developing seed at the beginning of the bulge (R5 stage); mature seed, seeds at maturity (R7 stage). This has been added in the illustration of Figure 2.

 

Discussion.

6. It was found that GmATX1 includes the conserved metal-binding sequence MXCXXC (Figure 1A), In addition, GmATX1 was found to be specifically localized to the nucleus and cell membrane (Figure 1B). The qRT-PCR results showed that the expression of GmATX1 was increased after HTH stress and heavy metal treatment, indicating that GmATX1 was indeed involved in the response to HTH stress and heavy metal treatment in soybean (Figure 2ABEF). Overexpression of GmATX1 in Arabidopsis enhanced developing seed viability (Figure 3AB) and seedling tolerance under HT and HTH stresses (Figure 3CDE). The results were confirmed by the silence of GmATX1 (Figure 4C). – lines 272-278, very short discussion regarding GmATX1 expression in various tissues and under different stresses in soybean, enhancement of seed vitality and seedling tolerance in soybean and GmATX1-overexpressed Arabidopsis under stresses, GmATX1 enhancing antioxidase activity and ROS scavenging in soybean and transgenic Arabidopsis seedlings under stresses,

Answer: We have rewritten the Discussion. (see the Discussion)

 

7. No numbers in the references

Answer: We have corrected them.

 

8. Potted plants at R7 growth stage were subjected to HTH stress according to Wang et al. (2012) [28]. – lines 366-367. Wrong style of reference,

Answer: We have corrected it.

 

9. Antioxidase activity assay was performed according to Song et al. (2015) with some modifications [29]. --lines 412-413 Wrong style of reference

Answer: We have corrected it.

 

10. The level of lipid peroxidation was assessed by measuring malondialdehyde (MDA) content according to Chu et al. (2012) with minor modifications [30]. --lines 418-420, Wrong style of reference

Answer: We have corrected it.

 

11. H₂O₂ staining [31] was conducted according to Mao et al. (2015) using leaves of seedlings of the WT, GmATX1-overexpressed Arabidopsis lines (L3) and silent soybean lines (pTRV2-GmATX1) under the HH treatment (23 ℃, 90% RH), HT treatment (40℃, 70% RH) and HTH treatment (40℃, 90% RH) for 0 d and 2 d, respectively. --lines 425-428, Wrong style of reference

Answer: We have corrected it.

 

12. ROS levels [32] were measured as previously described by Liu et al. (2017) using harvested seeds of the WT and GmATX1-overexpressed Arabidopsis lines (L3) under the HH, HT and HTH treatments for 0 d and 2 d, respectively. --lines 430-433 Wrong style of reference

Answer: We have corrected it.

Reviewer 2 Report

The manuscript no plants-1681426 titled: “GmATX1 promotes seed vigor and seedling tolerance under heavy metal and high temperature and humidity stresses” concerns the role of copper chaperone protein in stress . The experiment has been widely conducted using two plant species in different growth phases and different stress factors.

Unfortunately, the use of various research plant material for different analyzes made it difficult to clearly present the results. The authors usually make sure to specify what plant species the presented figure or description relates to, but describing Arabidopsis and soybean alternately gives the impression of chaos. Additionally, there are also some results for tobacco. It is why I suggest to rewrite the manuscript so that the description of one species is presented first, and then the other.

Figure 4 should be divided into two – one concerning Arabidopsis and the other concerning soybean.

The title should be more specify – I mean it should contain the full name of the studied protein and the species of plants. Therefore, it should sound for example:  The copper chaperone protein GmATX1 promotes seed vigor and seedling tolerance of Arabidopsis and soybean under heavy metal and high temperature and humidity stresses.

Figure 1B. Explain what pA7GmATX1::GFP and pA7::GFP mean. What is BF?

Figure 2C. The graphs show the GmATX1 expression in control plants (not treated by any stress)?

** indicates significant differences at p < 0.01. In comparison to control objects? Please specify.

Line 140. There is: more dark red seeds were observed. Please explain what it means, write rather that more seeds showed to be viable. How seed viability was determined is described in the methods.

By the way, there is lack of numerical data showing the results of seed viability test. The photos on Figure 3B are indicative only and should be replaced with a graph or a table.

Figure 3E. What units are on axis Y?

In case of the use of 50µM CuS0₄ there was no increased expression of GmATX1 gene in soybean but the same concentration of copper influences the root growth in Arabidopsis. The authors should discuss it in Discussion.

There is: “In our previous proteomics study, a copper chaperone protein gene, named GmATX1 was isolated from soybean” (line 264) and “in the present study, GmATX1 was isolated and characterized from soybean” (line 268). It made me confused. Additionally, the isolation of GmATX1 gene from soybean is not clearly presented in Materials and methods. There is only very short description which could indicate that the isolation of the gene was made in a previous study but there is not any reference given. In turn, if it was made in this study, the description of the procedure should be described in more detail. Please write clearly when the isolation was made, describe it or give reference.

Lines 264-265. The sentence is unclear and should be rephrased.

Lines 277-279. The statement is confusing. The authors compared the results of analyzes obtained for Arabidopsis and for soybean, but did not write that they studied gene silencing in soybean, which is misleading for the reader. Moreover, in my opinion, that the statement that a test result obtained for one species has been confirmed by testing another species is an abuse.

Although the chapter discussion presents the results of own research and that of other authors, the authors do not try to compare them. There is also no attempt to explain the mechanism by which changes in the activity of the studied gene lead to changes in stress resistance.

There is nothing in the Discussion about seed germination and viability.

The authors conclude that high temperature stress alone and high temperature combined with high humidity induced changes in gene activity and stress resistance. But the stress of HH didn't make any difference. So did the humidity really matter? Maybe in the case of HTH stress, only the heat mattered. Please consider it in Discussion.

As it stands, the discussion is more like a summary of the results and therefore needs to be improved.

Materials and methods, like Results, gives the impression of chaos caused by the use of several species. For example in subsection 4.2 it is not clear what plants RNA was isolated from.

Line 334. Please, explain why kanamycin was used.

Line 349 and 359. What is GmPDS?

Line 366. What do mean as “normally maintained ”? Please, specify.

Line 375. 10 plants within each treatment (HT, HH, HTH)? Please, specify.

Line 379. Which soybean seeds were germinated? Please, specify.

Line 389=390. What about light conditions in case of stressed seeds?

Chapter 5 is wrongly named as Discussion. It is summary.

Line 444 - 446. Please specify which species this statement applies to.

References. In text references to source articles are given as numbers while the reference list is prepared without numbering.

Author Response

Dear Reviewer:

 

Thank you for your valuable and thoughtful comments and suggestions for our manuscript. We appreciate your excellent comments and suggestions. We have considered all of these suggestions and comments in revising our manuscript. The following lists our responses to your comments.

 

Best Wishes,

 

Hao Ma

 

State Key Lab of Crop Genetics and Germplasm Enhancement,

Nanjing Agricultural University,

Nanjing, Jiangsu Province 210095, China

 

 

Answer to the reviewers’ comments:

 

1. Unfortunately, the use of various research plant material for different analyzes made it difficult to clearly present the results. The authors usually make sure to specify what plant species the presented figure or description relates to, but describing Arabidopsis and soybean alternately gives the impression of chaos. Additionally, there are also some results for tobacco. It is why I suggest to rewrite the manuscript so that the description of one species is presented first, and then the other.

Answer: We have rewritten the whole manuscript according to your valuable and thoughtful suggestion.

 

2. Figure 4 should be divided into two – one concerning Arabidopsis and the other concerning soybean.

Answer: We have divided into two.

 

3. The title should be more specify – I mean it should contain the full name of the studied protein and the species of plants. Therefore, it should sound for example:  The copper chaperone protein GmATX1 promotes seed vigor and seedling tolerance of Arabidopsis and soybean under heavy metal and high temperature and humidity stresses.

Answer: We have modified it.

 

4. Figure 1B. Explain what pA7GmATX1::GFP and pA7::GFP mean. What is BF?

Answer: We have added the explanation in the illustration of Figure 1.

 

5. Figure 2C. The graphs show the GmATX1 expression in control plants (not treated by any stress)?

Answer: The graphs show the GmATX1 expression in various tissues under normal growth and development.  

 

6. ** indicates significant differences at p < 0.01. In comparison to control objects? Please specify.

Answer: We have rewritten all these sentences.

 

7. Line 140. There is: more dark red seeds were observed. Please explain what it means, write rather that more seeds showed to be viable. How seed viability was determined is described in the methods.

Answer: We have rewritten the sentence. How seed viability was determined has been described in the methods.

 

8. By the way, there is lack of numerical data showing the results of seed viability test. The photos on Figure 3B are indicative only and should be replaced with a graph or a table.

Answer: We have added two tables.

 

9. Figure 3E. What units are on axis Y?

Answer: the unit has been added.

 

10. In case of the use of 50µM CuS04 there was no increased expression of GmATX1 gene in soybean but the same concentration of copper influences the root growth in Arabidopsis. The authors should discuss it in Discussion.

Answer: We have added the discussion in Discussion.

 

11. There is: “In our previous proteomics study, a copper chaperone protein gene, named GmATX1 was isolated from soybean” (line 264) and “in the present study, GmATX1 was isolated and characterized from soybean” (line 268). It made me confused. Additionally, the isolation of GmATX1 gene from soybean is not clearly presented in Materials and methods. There is only very short description which could indicate that the isolation of the gene was made in a previous study but there is not any reference given. In turn, if it was made in this study, the description of the procedure should be described in more detail. Please write clearly when the isolation was made, describe it or give reference.

Answer: We have corrected the related sentences to avoid the confusion. And the isolation of GmATX1 gene was described in more detail in Materials and Methods.

 

12. Lines 264-265. The sentence is unclear and should be rephrased.

Answer: We have rephrased the sentence.

 

13. Lines 277-279. The statement is confusing. The authors compared the results of analyzes obtained for Arabidopsis and for soybean, but did not write that they studied gene silencing in soybean, which is misleading for the reader. Moreover, in my opinion, that the statement that a test result obtained for one species has been confirmed by testing another species is an abuse.

Answer: We have rewritten them.

 

14. Although the chapter discussion presents the results of own research and that of other authors, the authors do not try to compare them. There is also no attempt to explain the mechanism by which changes in the activity of the studied gene lead to changes in stress resistance.

Answer: We have rewritten the whole Discussion and try to discuss the mechanism.

 

15. There is nothing in the Discussion about seed germination and viability.

Answer: We have rewritten the whole Discussion and added the discussion about seed germination and viability.

 

16. The authors conclude that high temperature stress alone and high temperature combined with high humidity induced changes in gene activity and stress resistance. But the stress of HH didn't make any difference. So did the humidity really matter? Maybe in the case of HTH stress, only the heat mattered. Please consider it in Discussion.

Answer: We have added discussion about this in the Discussion.

 

17. As it stands, the discussion is more like a summary of the results and therefore needs to be improved.

Answer: We have rewritten the Discussion.

 

18. Materials and methods, like Results, gives the impression of chaos caused by the use of several species. For example in subsection 4.2 it is not clear what plants RNA was isolated from.

Answer: We have rewritten the whole manuscript.

 

19. Line 334. Please, explain why kanamycin was used.

Answer: Because the constructed overexpression vector pBI121 is Kana resistant and wild-type seeds do not grow normally on kana medium.

 

20. Line 349 and 359. What is GmPDS?

Answer: Phytoene desaturase gene (PDS) is a key enzyme for carotenoid synthesis in plants. When PDS expression was suppressed, carotenoid content was significantly reduced and plant leaves appeared albino, and the PDS gene could be used as an indicator gene to detect whether the infested plant genes had a silent trend. The PDS gene can be used as an indicator gene to detect whether the infested plant genes have a silent trend.

 

21. Line 366. What do mean as “normally maintained”? Please, specify.

Answer: the sentence has been modified.

 

22. Line 375. 10 plants within each treatment (HT, HH, HTH)? Please, specify.

Answer: we have specified them.

 

23. Line 379. Which soybean seeds were germinated? Please, specify.

Answer: we have specified them in supplementary materials.

 

24. Line 389=390. What about light conditions in case of stressed seeds?

Answer: we have specified them.

 

25. Chapter 5 is wrongly named as Discussion. It is summary.

Answer: We have corrected.

 

26. Line 444 - 446. Please specify which species this statement applies to.

Answer: we have specified them.

Reviewer 3 Report

Revision to

 

Title: GmATX1 promotes seed vigor and seedling tolerance under heavy metal and high temperature and humidity stresses

 

Authors: Yingzi Shen, Jiaping Wei, Shuang Wang, Xi Zhang, Kebing Mu, Sushuang Liu, Hao Ma.

 

Abstract Journal: Plants

 

Manuscript number: plants-1681426

 

General remarks: The manuscript by Shen et al. investigate the role of GmATX1 a copper chaperone protein. The authors characterized the roles of this protein in a published paper by proteomics approach. On the other hand, the present manuscript reported an expression analysis of GmATX1 in soybean both in different tissues both upon humidity and heavy metals stress (Cu and Cd). Furthermore, the authors obtained an GmATX1 overexpressing Arabidopsis genotype and an Arabidopsis knock out for this gene. These genotypes were analyzed upon heat stress, humidity stress and in a combination of these.

The data would be interesting giving important information about this gene but the manuscript is not appropriately written. A significant improvement of the English language is mandatory for the final acceptance. Particularly the use of a Professional editing service or a revision by a native speaker is required.

The two part of the manuscript appear not quite connected. Particularly, the expression analysis section looks as a repetition of the previous paper published by the authors. The results and discussion sections are not described in detail and the quality of presentation is confused. Particularly the discussion is too short giving no new insight about the topic and no dissections about the arguments of the authors. The manuscript should be fully restructured. Another important question is: why the authors adopted the specific temperatures, humidity and heavy metals treatments described in this manuscript for soybean and Arabidopsis? Authors should give a reasonable basis for such selections.

Based on these considerations, the present version of this manuscript is rejected for publication on Plants. Anyway the author have at their disposal all the results and the means to reorganize and correct  the manuscript, and then resubmit it.  

 

Abstract:

Line 14 Please avoid the repetition of “proteins”

Line 17 Please verify that orthodoxies is the appropriate term

Lines 21-29 This part of the abstract is confusingly written, please rewrite.

 

Introduction:

Please detailed the importance of Soybean and the effects of abiotic stresses on this crop.

Line 72 – Please add references.

 

Results:

Authors should improve the results section improving the quantitative information. Furthermore, Authors should better underline the effects of different treatments, because this will reinforce their data and their arguments about the importance of GmATX1.

 

 

Methods:

Line 326 – Please cite the housekeeping gene for qRT-PCR.

 

References:

MDPI guidelines require numbered references. Authors numbered references in the main text but these in the reference list are in alphabetical order.

 

 

Minor points:

Authors selected “Cu” as abbreviation for copper. This term should be mentioned the first time only. Then the authors should use “Cu”. An exception is represented by the family of chaperones.

 

Authors should replace “Discussion” with “Conclusions” in section 5.

 

A lot of terms were incorrectly written, please revise.

Author Response

Dear Reviewer:

 

Thank you for your valuable and thoughtful comments and suggestions for our manuscript. We appreciate your excellent comments and suggestions. We have considered all of these suggestions and comments in revising our manuscript. The following lists our responses to your comments.

 

Best Wishes,

 

Hao Ma

 

State Key Lab of Crop Genetics and Germplasm Enhancement,

Nanjing Agricultural University,

Nanjing, Jiangsu Province 210095, China

 

 

Answer to the reviewers’ comments:

 

1. Why the authors adopted the specific temperatures, humidity and heavy metals treatments described in this manuscript for soybean and Arabidopsis? Authors should give a reasonable basis for such selections.

Answer: We have rewritten the whole manuscript. The research on heavy metals has been put into Discussion. The whole manuscript was focused on HTH stress, with HT and HH as control.

 

Abstract:

 

2. Line 14 Please avoid the repetition of “proteins”

Answer: We have corrected.

 

3. Line 17 Please verify that orthodoxies is the appropriate term

Answer: We have corrected the error.

 

4. Lines 21-29 This part of the abstract is confusingly written, please rewrite

Answer: We have rewritten them.

 

Introduction:

 

5. Please detailed the importance of Soybean and the effects of abiotic stresses on this crop.

Answer: we have detailed the importance of soybean and the effects of abiotic stresses on this crop.

 

6. Line 72 Please add references.

Answer: we have added the reference.

 

Results:

 

7. Authors should improve the results section improving the quantitative information. Furthermore, Authors should better underline the effects of different treatments, because this will reinforce their data and their arguments about the importance of GmATX1

Answer: We have rewritten the results section and added two tables.

 

Methods:

 

8. Line 326, Please cite the housekeeping gene for qRT-PCR.

Answer: we have added the housekeeping gene for qRT-PCR.

 

References:

 

9. MDPI guidelines require numbered references. Authors numbered references in the main text but these in the reference list are in alphabetical order.

Answer: We have numbered references.

 

Minor points:

 

10. Authors selected “Cu” as abbreviation for copper. This term should be mentioned the first time only. Then the authors should use “Cu”. An exception is represented by the family of chaperones.

Answer: We have corrected them.

 

11. Authors should replace “Discussion” with “Conclusions” in section 5.

Answer: We have corrected it.

 

12. A lot of terms were incorrectly written, please revise.

Answer: We have check carefully and corrected them.

Round 2

Reviewer 2 Report

I would like to thank the authors for their response and many corrections in the manuscript. Unfortunately, not all of my suggestions have been included in the text. Therefore, I still have some comments.  

In the title still there are no the names of plant species which were research material. The author should specify which species the article concerns.

The names of genes should be italicized while the names of proteins should not be italicized. The authors must look through the whole manuscript and correct it.

The authors use the phrase “seed growth” which is incorrect. They should look through the whole manuscript and remove this phrase.

Figure 2. The authors should precise the subtitle for C and D that the presented results concerns plants which were not subjected to any stress.

I do not understand why the authors removed the fragment about the influence of Cu and Cd. Even if they decided to present the figures as supplemental material (instead of in manuscript body), they should be commented in Result section and the methods of this experiment should be described in Material and Methods.

Line 181-182 There is: Moreover, the stomatal opening of the WT plants was nearly completely closed… ” Please, rewrite the sentence. It is understandable but “opening which is completely closed” do not sound good.

Tables 1 and 2. In the subtitles the authors should explain what are HH, HT and HTH.

I suggest to change the title of table 2 into: Viability of seeds  (%) of GmATX1-overexpression Arabidopsis lines (L1 and L2) and WT after different treatments.

Line 234. The authors mentioned their previous study (there is “In our previous proteomics study…”) therefore they should cite the reference where the study is presented.

Line 243-244. There is “…both during development and under HTH stress.” What do the authors mean as “during development”? development of seeds not subjected to any stress? It must be clearly written in the manuscript.

Line 326. It should be: 50 μmol/L CdCl₂

Line 340. There is “photoperiod of 16 h”. 16 h of light? The authors should precise.

Line 390. The authors should explain in the manuscript why media with kanamycin was used. Please explain that the used transgene contained selectable marker gene which confers resistance to kanamycin.

Line 405. There is: 10 treated plants of two soybean cultivars. I suggest to specify: 10 treated plants of each soybean cultivars.

Line 418. I suggest to change “photostage” into “photoperiod”.

Line 438. I suggest to move the reference and place it just after the author’s name. It should be “Song et al. [37] with some modifications”

Line 450. I suggest to place the reference just after the author’s name. It should be “Mao et al. [39] using the leaves…”

I strongly suggest also that the authors should make a professional linguistic correction because there are grammatical errors and many stylistic ones in the text. 

Author Response

Dear Reviewer:

 

Thank you for your valuable and thoughtful comments and suggestions for our manuscript. We appreciate your excellent comments and suggestions. We have considered all of these suggestions and comments in revising our manuscript. The following lists our responses to your comments.

 

Best Wishes,

 

Hao Ma

 

State Key Lab of Crop Genetics and Germplasm Enhancement,

Nanjing Agricultural University,

Nanjing, Jiangsu Province 210095, China

 

 

Answer to the reviewers’ comments:

 

1. In the title still there are no the names of plant species which were research material. The author should specify which species the article concerns.

Answer: We have modified it.

 

2. The names of genes should be italicized while the names of proteins should not be italicized. The authors must look through the whole manuscript and correct it.

Answer: We have checked and modified it through the whole manuscript.

 

3. The authors use the phrase “seed growth” which is incorrect. They should look through the whole manuscript and remove this phrase.

Answer: We have modified it through the whole manuscript.

 

4. Figure 2. The authors should precise the subtitle for C and D that the presented results concerns plants which were not subjected to any stress.

Answer: We have modified it.

 

5. I do not understand why the authors removed the fragment about the influence of Cu and Cd. Even if they decided to present the figures as supplemental material (instead of in manuscript body), they should be commented in Result section and the methods of this experiment should be described in Material and Methods.

Answer: We have added them in the Results.

 

6. Line 181-182 There is: Moreover, the stomatal opening of the WT plants was nearly completely closed… ” Please, rewrite the sentence. It is understandable but “opening which is completely closed” do not sound good.

Answer: We have modified it.

 

7. Tables 1 and 2. In the subtitles the authors should explain what are HH, HT and HTH.

Answer: We have added the explanation in the Tables 1 and 2.

 

8. I suggest to change the title of table 2 into: Viability of seeds (%) of GmATX1-overexpression Arabidopsis lines (L1 and L2) and WT after different treatments

Answer: We have modified it.

 

9. Line 234. The authors mentioned their previous study (there is “In our previous proteomics study…”) therefore they should cite the reference where the study is presented.

Answer: We have added the reference.

 

10. Line 243-244. There is “…both during development and under HTH stress.” What do the authors mean as “during development”? development of seeds not subjected to any stress? It must be clearly written in the manuscript.

Answer: We have modified it.

 

11. Line 326. It should be: 50 μmol/L CdCl2

Answer: We have corrected it.

 

12. Line 340. There is “photoperiod of 16 h”. 16 h of light? The authors should precise

Answer: We have modified it.

 

13. Line 390. The authors should explain in the manuscript why media with kanamycin was used. Please explain that the used transgene contained selectable marker gene which confers resistance to kanamycin.

Answer: We have modified the sentence.

 

14. Line 405. There is: 10 treated plants of two soybean cultivars. I suggest to specify: 10 treated plants of each soybean cultivars.

Answer: We have modified it.

 

15. Line 418. I suggest to change “photostage” into “photoperiod”.

Answer: We have modified it.

 

16. Line 438. I suggest to move the reference and place it just after the author’s name. It should be “Song et al. [37] with some modifications”

Answer: We have modified it.

 

17. Line 450. I suggest to place the reference just after the author’s name. It should be “Mao et al. [39] using the leaves…”

Answer: We have modified it.

 

18. I strongly suggest also that the authors should make a professional linguistic correction because there are grammatical errors and many stylistic ones in the text.

Answer: We have check carefully and corrected them.

Reviewer 3 Report

The R1 version of the manuscript by Shen et al. is significantly improved. Particularly, English language and quality of presentation are now appropriate for a scientific manuscript. The manuscript is suitable for pubblication on Plants

Author Response

Thanks.