Specific Gibberellin 2-Oxidase 3 (SbGA2ox3) Mutants Promote Yield and Stress Tolerance in Sorghum bicolor
Round 1
Reviewer 1 Report
ABSTRACT
The abstract needs to be re-written. There are just too many errors, both in language and the scientific reporting. For example, why was EMS used in the study? Why random mutagenesis?
INTRODUCTION
1. The authors need to review literature of EMS as a mutagen and also why use it.
2. Line 40-41: please restructure the sentence
3. Line 43: do the authors mean that sorghum is the second most produced cereal worldwide? I think this incorrect. Please double check the production statistics of FAO stats.
4. Line 45: The word “second” is misplaced. Please delete.
5. Line 51-55. Please restructure the sentences. It is not clear to me why and how sorghum has attracted scholars? Also, it is not clear to me what quality means in these sentences.
6. Lines 56-58 need revision. What resistance is being referred to here? Furthermore, sentence construction in the lines need revision.
7. The abbreviation GA needs to be defined in the Introduction.
8. The tenses in some part of the introduction need to be corrected. For example, the use of “were” in lines 60 and 63 is incorrect.
9. Also, correct grammar e.g. when referring to multiple enzymes, then the sentence has to be corrected as such and vice versa. For example, in Line 66, is GA2oxs referring to multiple enzymes? Is yes, then the sentence should read GA2oxs function ….
10. Line 68: what does it mean the enzyme has been cloned and identified in a variety of plants? does this mean that the enzyme is not naturally occurring? Please correct.
11. All scientific names should be italisized. E.g. Arabidopsis thaliana in Line 69.
12. I also suggest that plants should be written both in their common and scientific names at first mention. Thereafter the common name can be used.
13. In Line 70, delete etc.
14. Lines 70-74. I do not understand this sentence. is this a natural mutation or what? and how do we know it is a natural mutation?
15. Line 75: what is StGA2ox1?
16. Line 76: what is meant by exogenous hormones?
17. In Line 77: what does expression mean? Is this over-expression or what?
18. In lines 79-81: I do not understand how GA2oxs comes into discussion here.
19. Lines 84-85 need revision.
MATERIALS & METHODS
1. The methods should be rewritten in such a way that they are repeatable by others.
2. In line 96, please add the word variety after sorghum.
3. In line 96, what is Guizhou Maotai?
4. Please state the country where the breeding center is located (line 97).
5. How did the authors select the mutagenic chemical EMS, the concentration to use and also the duration of chemical treatment? If the procedure what obtained from another publication, please cite the ref, otherwise justification of the above needs to be given.
6. Line 110: sowed should be written as “sown”. Reared should be written as “grown”.
7. Line 111 and elsewhere in the methods section: what is one heart?
8. Line 110: What are the growth conditions in the greenhouse?
9. Line 111: What are the growth conditions in the field?
10. Lines 123-126. I am not sure what this is about.
11. Line 131: the PCR procedure is shown……
12. Line 133: you need to define what SbGA2o3 is.
13. In section 2.6, it is not clear to me how drought stress was imposed, for how long etc.
14. Line 155: the subsection title should be Gene expression analysis of SbGA2ox3 in sorghum
15. Line 162: I would replace the term “non-biological stresses” with “abiotic stresses”.
16. In Lines 168-172, what type of PCR machine was used?
RESULTS
1. The quality of results presentation is good. Below are few comments.
2. Lines 179-180 should be written as one sentence without unnecessary repetition
3. Why is the sorghum variety ideal for EMS treatment? Line 181
4. Line 183: what aspects are being referred to?
5. Lines 185; the words “it will” need to be changed to a more cautious tone.
6. What are non-adversity resistance? Line 188
7. Line 193: delete “in detail”
8. Line 194, is the “normal sorghum Hongyingzi” the same as wild type? If yes, this needs to be clarified here as well as in the materials and methods section.
9. Line 194-195: is it Table 4 or Table 1?
10. Line 211: what is meant by “related to the sorghum plant type”?
11. What are nine-dwelling plants? Line 212
12. What do you mean by “accidentally branched at the test site” Line 214
13. Line 222: delete sea
14. Also check tenses in the results section, e.g. are in line 224, is in Line 268
15. Please revise line 225-226
16. Figure 2 needs to be revised: I would suggest having the WT plant as A, followed by all other phenotypes in B, C etc.
17. Higher and higher should just be written as higher in Line 288
18. The word “hundreds” in line 302-303 is inappropriate because none of the expression levels except 130X. please correct.
19. Line 340-342: the study did not look at ROS accumulation, so this sentence is inappropriate.
20. Line 342 is also very vague. Please correct.
21. Line 344: replace paper with study
22. I would delete the word strong; and replace resistance with tolerance
23. In figure 6, please recheck results if there are statistically significant differences in Figure 6 A & B. in the same figure, CK should be written in the same format in C and D.
DISCUSSION
While the discussion is well written the authors need to define the species of the different genes. E.g. ScGA2ox3, AaA2ox3 etc.
Two major coments for this research are:
1. why did the authors only look at one gene? Why not many genes even if they are isoforms of the one they looked at?
2. Furthermore, how certain are we that the phenotypic traits observed are due to the GAox and not other mutated genes?
3. Language use needs to be improved especially for the Abstract right up to the Results section.
Author Response
The abstract needs to be rewritten. There are just too many errors, both in language and scientific reporting. For example, why was EMS used in the study? Why random mutagenesis?
Response: Thank you very much for your advice. Modified in summary section.
INTRODUCTION
- The authors need to review the literature on EMS as a mutagen and also why use it.
Response: Thank you very much for your advice. This section has been added in the introduction.
- Line 40-41: please restructure the sentence
Response: Thank you very much for your advice. I've adjusted the sentence in the text.
- Line 43: do the authors mean that sorghum is the second most-produced cereal worldwide? I think this is incorrect. Please double-check the production statistics of FAO stats.
Response: I'm sorry the author has bothered you. The author means that sorghum is a cereal crop next to corn, rice, wheat, and barley. Sorghum is the fifth largest grain crop.
- Line 45: The word “second” is misplaced. Please delete.
Response: Thank you very much for your advice. This article changed from second to next.
- Line 51-55. Please restructure the sentences. It is not clear to me why and how sorghum has attracted scholars? Also, it is not clear to me what quality means in these sentences.
Response: (1) Thank you very much for your advice. Sentences have been remodification in the article. (2) Sorghum has attracted much attention due to its multiple uses and its economic benefits in the wine industry. (3) High-quality germplasm for different materials used in different standards: for example, wine sorghum, which is mainly used for brewing liquor, generally requires high starch content, appropriate protein, and tannin content, therefore, screening of high starch content, strong resistance varieties is the screening of high-quality varieties.
- Lines 56-58 need revision. What resistance is being referred to here? Furthermore, sentence construction in the lines needs revision.
Response: (1) Thank you very much for your advice. Resistance here refers to the ability to resist biotic and abiotic stresses. (2) The sentence has been modified.
- The abbreviation GA needs to be defined in the Introduction.
Response: Thank you very much for your advice. GA has been defined herein.
- The tenses in some parts of the introduction need to be corrected. For example, the use of “were” in lines 60 and 63 is incorrect.
Response: Thank you very much for your advice. Changed “were” to “are” in the text.
- Also, correct grammar e. g. when referring to multiple enzymes, then the sentence has to be corrected as such and vice versa. For example, in Line 66, is GA2oxs referring to multiple enzymes? Is yes, then the sentence should read GA2oxs function ….
Response: Thank you very much for your advice. GA2oxs is a type of enzyme.
- Line 68: what does it mean the enzyme has been cloned and identified in a variety of plants? Does this mean that the enzyme is not naturally occurring? Please correct.
Response: Thank you very much for your advice. Genes encoding this enzyme have been cloned and identified in a variety of plant species. This enzyme is present in plants.
- All scientific names should be italicized. E. g. Arabidopsis thaliana in Line 69.
Response: Thank you very much for your advice. Has been revised in the text.
- I also suggest that plants should be written both in their common and scientific names at first mention. Thereafter the common name can be used.
Response: Thank you very much for your advice. Modifications have been made herein.
- In Line 70, delete etc.
Response: Thank you very much for your advice. Has been deleted from the paper.
- Lines 70-74. I do not understand this sentence. is this a natural mutation or what? and how do we know it is a natural mutation?
Response: Thank you very much for your advice. The mutations referred to herein are point mutations and have been modified herein.
- Line 75: what is StGA2ox1?
Response: Thank you very much for your advice. It has been confirmed in the literature that the GhGA2ox1 gene in upland cotton was mentioned in this paper.
- Line 76: what is meant by exogenous hormones?
Response: Thank you very much for your advice. Exogenous plant hormones refer to synthetic chemicals that act like plant hormones.
- In Line 77: what does expression mean? Is this over-expression or what?
Response: Thank you very much for your advice. This is over-expression.
- In lines 79-81: I do not understand how GA2oxs comes into the discussion here.
Response: Thank you very much for your advice. GA2oxs genes have been found to regulate plant development and enhance plant resistance in rice, upland cotton, and other plants. Therefore, we speculated that the sorghum GA2ox3 in the study could also have similar functions.
- Lines 84-85 need revision.
Response: Thank you very much for your advice. Modifications have been made herein.
MATERIALS & METHODS
- The methods should be rewritten in such a way that they are repeatable by others.
Response: Thank you very much for your advice. The methods have been rewritten.
- In line 96, please add the word variety after sorghum.
Response: Thank you very much for your advice. The word “variety” has been added to the text accordingly.
- In line 96, what is Guizhou Maotai?
Response: Thank you very much for your advice. Kweichow Moutai refers to Kweichow Moutai Group. Kweichow Moutai is a traditional specialty wine in China. It is one of the world's three famous distilled liquors along with British Scotch Whisky and France Cognac Bandy and is also one of China's three famous liquors "Maotai Wuliangye Jiannanchun". The main raw material for brewing Maotai wine is 'Hongyingzi'.
- Please state the country where the breeding center is located (line 97).
Response: Thank you very much for your advice. Modifications have been made herein. In this study, the special liquor-making sorghum variety 'Hongyingzi' was selected by the Fengyuan Organic Sorghum Breeding Center of Renhuai City, Zunyi City, Guizhou Province, China.
- How did the authors select the mutagenic chemical EMS, the concentration to use, and also the duration of chemical treatment? If the procedure what obtained from another publication, please cite the ref, otherwise justification of the above needs to be given.
Response: Thank you very much for your advice. The concentration and time of EMS used in this study were summarized from the previous work in our laboratory. It was found that the survival rate of M0 of sorghum in the field was close to 50%, so this treatment was selected.
- Line 110: sowed should be written as “sown”. Reared should be written as “grown”.
Response: Thank you very much for your advice. Have been modified in that corresponding position herein.
- Line 111 and elsewhere in the methods section: what is one heart?
Response: Thank you very much for your advice. This period is the period when plants grow two true leaves and a terminal bud.
- Line 110: What are the growth conditions in the greenhouse?
Response: Thank you very much for your advice. The greenhouse environment was maintained at 70–75% relative humidity, 16-h light/8-h dark-light cycle, and 22 °C.
- Line 111: What are the growth conditions in the field?
Response: Thank you very much for your advice. It is located at 106°27'18″ - 106°52' 30″ E and 26°11' 10″- 26°34' 00″ N. This area belongs to the subtropical monsoon humid climate, with obvious plateau climate characteristics, warm in winter and cool in summer, and the spring and autumn climate is changeable. Water resources are rich, light energy resources are less, light, heat, and water in the same season, the vertical climate difference is obvious. The annual average temperature is 14. 9℃ and the rainfall is 1178. 1 mm.
- Lines 123-126. I am not sure what this is about.
Response: Thank you very much for your advice. Supplementary Table S1 for the detection criteria of sorghum agronomic traits.
- Line 131: the PCR procedure is shown……
Response: Thank you very much for your advice. The PCR procedure was shown as follows: 94 ◦C for 5 min followed by 35 cycles of 95 ◦C for 30 s, 60 ◦C for 30 s, and 72 ◦C for 30s; then 72 ◦C for 5min and 16 ◦C for ∞.
- Line 133: you need to define what SbGA2o3 is.
Response: Thank you very much for your advice. SbGA2ox3 is an oxidase that functions to convert bioactive GA1 and GA4 into non-bioactive GA8 and GA34.
- In section 2.6, it is not clear to me how drought stress was imposed, for how long, etc.
Response: Thank you very much for your advice. Watering was stopped after the plants were grouted and field phenotypic identification was conducted after five days of continuous drought.
- Line 155: the subsection title should be Gene expression analysis of SbGA2ox3 in sorghum
Response: Thank you very much for your advice. Modifications have been made herein.
- Line 162: I would replace the term “non-biological stresses” with “abiotic stresses”.
Response: Thank you very much for your advice. Modifications have been made herein.
- In Lines 168-172, what type of qPCR machine was used?
Response: Thank you very much for your advice. All procedures were performed by the manufacturer's instructions (Bio-Rad, Hercules, CA, USA).
RESULTS
- The quality of the presentation of the results is good. Below are a few comments.
Response: Thank you for your approval.
- Lines 179-180 should be written as one sentence without unnecessary repetition.
Response: Thank you very much for your advice. The second sentence has been deleted from the text.
- Why is the sorghum variety ideal for EMS treatment? Line 181
Response: Thank you very much for your advice. As the ‘Hongyingzi’ has the characteristics of easy threshing, wide adaptability, drought resistance, barren resistance, strong stress resistance, and high yield, and can meet the requirements of the Kweichow Maotai-flavor liquor brewing process.
- Line 183: what aspects are being referred to?
Response: Thank you very much for your advice. All aspects refer to comprehensive traits, including agronomic traits and quality traits that meet the requirements of production, as well as a certain ability to resist stress.
- Lines 185; the words “it will” need to be changed to a more cautious tone.
Response: Thank you very much for your advice. Modifications have been made herein (line 260).
- What is non-adversity resistance? Line 188
Response: Thank you very much for your advice. Non-advisability resistance has been revised as force majeure to mainly express factors beyond human control, such as unsuitable climate, serious bird damage, and the non-fertility of the plant.
- Line 193: delete “in detail”
Response: Thank you very much for your advice. Deleted in text.
- Line 194, is the “normal sorghum Hongyingzi” the same as the wild type? If yes, this needs to be clarified here as well as in the materials and methods section.
Response: Thank you very much for your advice. The word " normal sorghum Hongyingzi " is amended here as " untreated normal sorghum Hongyingzi". The untreated Hongyingzi plants were used as the control.
- Line 194-195: is it Table 4 or Table 1?
Response: Thank you very much for your advice. It is Table 1.
- Line 211: what is meant by “related to the sorghum plant type”?
Response: Thank you very much for your advice. The plant type in this study mainly includes plant height, tillering, and branch. Some traits related to the sorghum plant type were plant height, tillering, and branch.
- What are nine-dwelling plants? Line 212
Response: Thank you very much for your advice. It is nine plants.
- What do you mean by “accidentally branched at the test site” Line 214
Response: Thank you very much for your advice. It is nine plants. Some plants were unexpectedly found to have branches in the test site.
- Line 222: delete sea
Response: Thank you very much for your advice. Deleted in text.
- Also check tenses in the results section, e.g. are in line 224, is in Line 268
Response: Thank you very much for your advice. Has been modified in the text.
- Please revise lines 225-226
Response: Thank you very much for your advice. Has been modified in the text.
- Figure 2 needs to be revised: I would suggest having the WT plant as A, followed by all other phenotypes in B, C, etc.
Response: Thank you very much for your advice. Has been modified in the text.
- Higher and higher should just be written as higher in Line 288
Response: Thank you very much for your advice. Has been modified in the text.
- The word “hundreds” in lines 302-303 is inappropriate because none of the expression levels except 130X. Please correct.
Response: Thank you very much for your advice. Has been modified in the text.
- Line 340-342: the study did not look at ROS accumulation, so this sentence is inappropriate.
Response: Thank you very much for your advice. Has been modified in the text.
- Line 342 is also very vague. Please correct.
Response: Thank you very much for your advice. Has been modified in the text.
- Line 344: replace paper with study
Response: Thank you very much for your advice. Has been modified in the text.
- I would delete the word strong; and replace resistance with tolerance
Response: Thank you very much for your advice. Has been modified in the text.
- In figure 6, please recheck results if there are statistically significant differences in Figure 6 A & B. in the same figure, CK should be written in the same format in C and D.
Response: Thank you very much for your advice. When detecting the induced expression levels of SbGA2ox3 under different abiotic stresses, tissues in the 0-hour treatment were used as the control, and the significance analysis was conducted separately for each treatment. Thus, the control criteria were consistent for each treatment. The differences between the different stress treatments were not discussed in this paper.
DISCUSSION
While the discussion is well written the authors need to define the species of the different genes. E. g. ScGA2ox3, AaGA2ox4 etc.
Response: Thank you very much for your advice. ScGA2ox3 has a writing error that has been corrected; AaGA2ox4 is a gene in a breadfruit tree and OsGA2ox6 is a gene in rice, all of which are described in the text as genes in what species.
Two major comments for this research are:
- Why did the authors only look at one gene? Why not many genes even if they are isoforms of the one they looked at?
Response: Thank you very much for your advice. A large number of excellent materials were created in this study, which laid a solid foundation for us to explore functional genes and improve varieties. Therefore, we will spend a large amount of energy on the utilization of excellent materials and the mining and verification of more functional genes.
- Furthermore, how certain are we that the phenotypic traits observed are due to the GA2ox and not other mutated genes?
GA2ox converts biologically active GA1 and GA4 to non-biologically active GA8 and GA34 mainly through hydroxylation of C-2. In this experiment, we observed that the mutant line had shorter plants, shorter leaves, and enhanced resistance, all of which were related to SbGA2ox3. Therefore, we selected this gene as the target gene. However, we need further proof next, what effect did the mutation of this gene have on the plant; Whether other genes besides the gene are related to the trait?
- Language use needs to be improved especially for the Abstract right up to the Results section.
Response: Thank you very much for your advice. We have polished up the text.
Reviewer 2 Report
In the manuscript the authors generated EMS mutants of sorghum. Next they characterized obtained mutant plants regards as several agronomic traits. They also focused on one of gene - gibberellin type II oxidase gene (SbGA2ox3), they analyzed the changes in the sequence within it.
The authors have already stated in the title that mutations in the gene SbGA2ox3 are the reasons for the increase in plant yield and stress tolerance. However, the mutation factor, which they used, is not selective but it generates mutations within whole genome. Therefore the changes of agronomic performance or stress response may be result of different mutations. Meanwhile, other mutations were not analyzed by the authors. Moreover, the authors studied expression of the gene SbGA2ox3 but it is not clear in which kind of plants (wild type, mutant?) There is something about sequencing of the gene in chapter 2.3 and then about expression in 2.7 but the plant material was not determined clearly enough. The authors write about stress tolerance while in fact they did not observe the tolerance but only changes in gene expression that occurred under different stress conditions. And this only proves that the tested gene reacts with a change in expression under stress conditions, but it does not prove that it is responsible for plant tolerance. Anyway, it is difficult to talk about tolerance since the authors do not present the results of plant response to stress.
The above concerns are crucial when it comes to assessing the quality of manuscript. The manuscript should be thoroughly redrafted.
There are also numerous other comments.
Line 112. How the plants were protected from uncontrolled crossing?
Line 128. The authors should describe the procedure of DNA extraction. It is too brief.
Lines 135-137. Unclear.
Lines 140-144. Do marks x, * and X mean the same or something different?
Lines 151-154. The description of a method too brief. What the authors mean as “drought in the field”? Any parameters? Any details?
Chapter 2.7 What plants of sorghum were used in that tests?
Lines 179-186. This fragment does not present results. It is good for introduction.
Line 194. What do the authors mean as “normal plats”? not mutated? wild-type?
Line 194. There is not table 4, maybe it should be table 1.
Line 217. What does it mean “(± 20 cm)” here?
Lines 225, 231. Where this data is shown in the results?
Lines 241 – 247. Unclear. Obtaining the mutants may broaden the germplasm resources and only their characterization can expand the mutant library.
Line 335. There is “ plant 3614 was less affected by drought” . Where it is shown that this plant was less affected by drought? Less than what?
Lines 340-343. Where these results are shown?
Line 335 and 344. 3614 is a number of a plant or a number of plants?
Lines 362 -367. These is general information. It would be good for introduction.
Line 374. Figure 1 shows mutations of spike.
Line 377- 382. Unclear.
Line 388. What is “biological yield”?
Line 389. What is “ineffective tillering”?
Line 397. What is “ content of agriculture”?
Line 423. 1/16.50kb Where it is in the results?
Lines 430-433. Unclear.
Lines 441-446. Unclear.
Chapter 3.2 and figure 4. This analyses is useless for the presented study.
The titles of the tables are not informative enough. The table should be understandable for a reader without having to look for an explanation in the text of the manuscript.
Figure 1. It would be better to divide clearly the photos marked as A, B, C and D.
Figures 5 and 6. What are letters a b c d over the bars?
Figure 7. What is ** over the bar?
Author Response
The authors have already stated in the title that mutations in the gene SbGA2ox3 are the reasons for the increase in plant yield and stress tolerance. However, the mutation factor, which they used, is not selective but it generates mutations within whole genome. Therefore the changes of agronomic performance or stress response may be result of different mutations. Meanwhile, other mutations were not analyzed by the authors. Moreover, the authors studied expression of the gene SbGA2ox3 but it is not clear in which kind of plants (wild type, mutant?) There is something about sequencing of the gene in chapter 2.3 and then about expression in 2.7 but the plant material was not determined clearly enough. The authors write about stress tolerance while in fact they did not observe the tolerance but only changes in gene expression that occurred under different stress conditions. And this only proves that the tested gene reacts with a change in expression under stress conditions, but it does not prove that it is responsible for plant tolerance. Anyway, it is difficult to talk about tolerance since the authors do not present the results of plant response to stress. The above concerns are crucial when it comes to assessing the quality of manuscript. The manuscript should be thoroughly redrafted.
There are also numerous other comments.
Response: Thank you very much for your advice. All mutation factors selected by us were determined through relevant literature and high-throughput sequencing technology. Agronomic traits and stress mutations are mostly controlled by poly genes; Therefore, we mainly wanted to show that SbGA2ox3 was one of the key genes for these traits. We studied the expression of the SbGA2ox3 gene in wild-type ‘Hongyingzi’. Through drought stress treatment, we selected the final mutant material, and we observed that it was less affected by drought stress compared with wild type, and detected the content of soluble sugar and MDA under drought stress to further illustrate the enhanced.
- Line 112. How the plants were protected from uncontrolled crossing?
Response: Thank you very much for your advice. All plants were bagged in this study.
- Line 128. The authors should describe the procedure of DNA extraction. It is too brief.
Response: Thank you very much for your advice.It has been modified in the text.
- Lines 135-137. Unclear.
Response: Thank you very much for your advice. It has been modified in that corresponding position herein.
- Lines 140-144. Do marks x, * and X mean the same or something different?
Response: Thank you very much for your advice. The ‘*’ and ‘X’ represent ‘x’, it has been modified in the corresponding position
- Lines 151-154. The description of a method too brief. What the authors mean as “drought in the field”? Any parameters? Any details?
Response: Thank you very much for your advice. After grain filling of sorghum, watering of the plants in the flowerpots was stopped, and the physiological indexes were detected after continuous drought for 5 days. Respectively weighing 0.1g of the sorghum leaves after the drought. The contents of soluble sugar and malondialdehyde in sorghum leaves subjected to drought treatment were determined according to the kit instructions produced by Beijing Bosch Biotechnology Co., Ltd.
- Chapter 2.7 What plants of sorghum were used in that tests?
Response: Thank you very much for your advice. The non-mutagenized Guizhou province local sorghum varieties ‘Hongyingzi’ was used in this test.
- Lines 179-186. This fragment does not present results. It is good for introduction.
Response: Thank you very much for your advice. This paragraph has been included in the introduction.
- Line 194. What do the authors mean as “normal plats”? not mutated? wild-type?
Response: Thank you very much for your advice. It meant no mutation.
- Line 194. There is not table 4, maybe it should be table 1.
Response: Thank you very much for your advice. It has been modified in the text.
- Line 217. What does it mean “(± 20 cm)” here?
Response: Thank you very much for your advice. Under the same cultivation conditions, the mutant plants were 20 cm taller or shorter than the non-mutagenized plants and were considered to be taller or shorter.
- 225, 231. Where this data is shown in the results?
Response: Thank you very much for your advice. These data are shown in table 1 in the results.
12.Lines 241 – 247. Unclear. Obtaining the mutants may broaden the germplasm resources and only their characterization can expand the mutant library.
Response: Thank you very much for your advice. It has been modified in the text.
- Line 335. There is “plant 3614 was less affected by drought” . Where it is shown that this plant was less affected by drought? Less than what?
Response: Thank you very much for your advice. As shown in Figure 7C and D, soluble sugar content of plant 3614 was significantly higher than that of wild-type plant and MDA content was significantly lower than that of wild-type plant after drought stress. Therefore, the damage of drought stress on plant 3614 was lower than that of wild type.
- Lines 340-343. Where these results are shown?
Response: Thank you very much for your advice. This part is not covered in this study, so this sentence has been deleted.
- Line 335 and 344. 3614 is a number of a plant or a number of plants?
Response: Thank you very much for your advice. 3614 is the number of the plant.
- 362 -367. This is general information. It would be good for introduction.
Response: Thank you very much for your advice. It has been modified in the text.
- Line 374. Figure 1 shows mutations of spike.
Response: Thank you very much for your advice. It has been modified in the text.
- Line 377- 382. Unclear.
Response: Thank you very much for your advice. Has been modified in the text.
- Line 388. What is “biological yield”?
Response: Thank you very much for your advice. Biological yield refers to the total amount of organic matter accumulated during the whole growth process of crops. Cereal crops such as rice and wheat are usually calculated based on above-ground yield; Sweet potato and potato are calculated based on the total yield of above-ground and underground.
- Line 389. What is “ineffective tillering”?
Response: Thank you very much for your advice. In the mature period can not heading or can heading and seed grain number less than 10 tillers, called ineffective tillers.
- Line 397. What is “content of agriculture”?
Response: Thank you very much for your advice. Has been modified in the text. “content of agriculture” refer to the objectives of agricultural production.
- Line 423. 1/16.50kb Where it is in the results?
Response: Thank you very much for your advice. It has been modified in the text.
- Lines 430-433. Unclear.
Response: Thank you very much for your advice. It has been modified in the text.
- Lines 441-446. Unclear.
Response: Thank you very much for your advice. It has been modified in the text.
- Chapter 3.2 and figure 4. This analyses is useless for the presented study.
Response: Thank you very much for your advice. In this paper, the structural prediction, physiological and biochemical analysis and phylogenetic tree construction of SbGA2ox3 are designed to let us understand more about SbGA2ox3, and that the SbGA2ox3 used in this study is really what we want.
- The titles of the tables are not informative enough. The table should be understandable for a reader without having to look for an explanation in the text of the manuscript.
Response: Thank you very much for your advice. It has been modified in the text.
- Figure 1. It would be better to divide clearly the photos marked as A, B, C and D.
Response: Thank you very much for your advice. 27. Figure 1 has been modified in the text.
- Figures 5 and 6. What are letters a b c d over the bars?
Response: Thank you very much for your advice. Significant differences are indicated between the different letters.
- Figure 7. What is ** over the bar?
Response: Thank you very much for your advice. It has been modified in the text.
Reviewer 3 Report
The overall merit of the paper is high: the problem presented in the introduction is quite relevant and the technological solutions explored by the paper are scientifically sound. The paper presents two major weaknesses: the description of the methods is not sufficiently accurate and the scientific teminology is not precise. The description of the mutation breeding experiment in particular is lacking many aspects: how many M0 seeds were treated with EMS? How were M1 plants harvested (in bulk, by spike, by tiller, by plant)? How were M2 lines grown (by population bulk system, by ear to row, by plant to row, etc. The method used for determination of drought resistance is not described at all. The data from morph-physiological analysis are not reported (what does "significantly shorter" mean?).
The terminology used is not very accurate:
line 188 non- adversity resistance (?)
line 318 accessions (mutant lines?)
line 357 Brewed sorghum (brewing sorghum?)
line 358 main parent trains included among problems (?)
line 361 genetic breeding (genetic improvement?)
line 397 main content of modern agriculture (?)
line 417 mutagenic population (mutagenised population?)
etc.....
The lines 179-180 and 379-380 do not make sense (is there a repetition?)
Author Response
1、The terminology used is not very accurate:
Response: Thank you very much for your advice. Has been modified in the text.
2、Line 188 non- adversity resistance (?)
Response: Thank you very much for your advice. Has been modified in the text.
3、Line 318 accessions (mutant lines?)
Response: Thank you very much for your advice. Yes, there are 415 sorghum mutant lines.
4、Line 357 Brewed sorghum (brewing sorghum?)
Response: Thank you very much for your advice. Yes, it is brewing sorghum.
5、Line 358 main parent trains included among problems (?)
Response: Thank you very much for your advice. I think it exists
6、Line 361 genetic breeding (genetic improvement?)
Response: Thank you very much for your advice. Has been modified in the text.
7、Line 397 main content of modern agriculture (?)
Response: Thank you very much for your advice. Has been modified in the text. ‘content of modern agriculture’ was rewritten as ‘objectives of agricultural production’.
8、Line 417 mutagenic population (mutagenized population?) etc.....
Response: Thank you very much for your advice. Has been modified in the text.
9、The lines 179-180 and 379-380 do not make sense (is there a repetition?)
Response: Thank you very much for your advice. Has been modified in the text.
Author Response File: 
Author Response.docx
Reviewer 4 Report
The purpose of the article “Specific Gibberellin 2-oxidase 3 (SbGA2ox3) mutants promote yield and stress tolerance in Sorghum bicolor” is reported a detailed to identify and characterize the sorghum mutagenized by EMS and an effective mutant bank was established to screen out mutant plants with variations in plant type, leaf shape, spike development, glume shell, growth period length, fertility, plant height, and drought resistance. Overall, findings of this study provide the results that the GA2ox gene mutant was screened out by PCR product sequencing, and its agronomic traits and stress resistance were identified. The article, however, must be improved in terms of writing since some grammar and syntax errors are present in the manuscript. They should address the subject and critically review the information from the literature.
My suggestions:
The abstract is written in a way that lacks logic. It should highlight the salient findings more critically.
Keywords are present in the title (Sorghum bicolor; SbGA2ox3; stress tolerance), choose others.
Introduction needs a more convincing rationale for this article, has short paragraphs, and I suggest incrementing the size of the paragraphs and text. The introduction does say little about the selected Sorghum bicolor, why were these selected?
Provide experimental work plans at the start of M&M. No detailed description is available about the experiment.
The report on M&M is very succinct! I suggest a better description of the experiments carried out, such as: Investigation of the agronomic character of plants in the field; Mutation Density Evaluation; SbGA2ox3 Sequences and Phylogenetic Analysis; Determination of soluble sugar and malondialdehyde content; and The expression analysis of SbGA2ox3 in sorghum;
The results have short paragraphs, I suggest incrementing the size of the paragraphs and text. Authors should discuss the results integrally. The discussion is based on individual results. I suggest that integrating the results will give more value to the work. I suggest that you discuss by integrating all your results (Pearson correlation or PCA).
Figure 1, 2 and 3: Please provide scale bars.
The results of this study are not fully explained therefore the interpretation of the results is very difficult. The author needs to provide the % increase or decrease rather than just writing ''significantly increased….''.
The discussion is poorly written hence, needs rewriting. The discussion should be further strengthened by adding some more relevant papers. The literature search is insufficient, only few related research papers in the past five years are cited (31,57%), and add the latest research results appropriately. See the links below if you think it will benefit your discussion.
Author Response
- The abstract is written in a way that lacks logic. It should highlight the salient findings more critically.
Response: Thank you very much for your advice. Abstract has been modified in the text.
- Keywords are present in the title (Sorghum bicolor; SbGA2ox3; stress tolerance), choose others.
Response: Thank you very much for your advice. Keywords have been modified in the text.
- Introduction needs a more convincing rationale for this article, has short paragraphs, and I suggest incrementing the size of the paragraphs and text. The introduction does say little about the selected Sorghum bicolor, why were these selected?
Response: Thank you very much for your advice. Introduction has been modified in the text.
- Provide experimental work plans at the start of M&M. No detailed description is available about the experiment.
Response: Thank you very much for your advice. M&M have been modified in the text.
- The report on M&M is very succinct! I suggest a better description of the experiments carried out, such as: Investigation of the agronomic character of plants in the field; Mutation Density Evaluation; SbGA2ox3 Sequences and Phylogenetic Analysis; Determination of soluble sugar and malondialdehyde content; and the expression analysis of SbGA2ox3 in sorghum;
Response: Thank you very much for your advice. M&M have been modified in the text.
- The results have short paragraphs; I suggest incrementing the size of the paragraphs and text. Authors should discuss the results integrally. The discussion is based on individual results. I suggest that integrating the results will give more value to the work. I suggest that you discuss by integrating all your results (Pearson correlation or PCA).
Response: Thank you very much for your advice.The main purpose of this study was to create useful mutant materials for sorghum germplasm resources and sorghum breeding. The EMS mutation is an undirected mutation. To mutagenize sorghum materials with EMS is to obtain some mutations that are difficult to obtain under the natural mutation conditions. From the results of this study, we have abundant mutation types in our mutant library, and most of them are worthy of study. In order to cultivate more abundant sorghum varieties, each type is worth studying, so we discussed each type, to lay the foundation for future breeding.
- Figure 1, 2 and 3: Please provide scale bars.
Response: We have provided scale bars in Figure 1, 2 and 3.
- The results of this study are not fully explained therefore the interpretation of the results is very difficult. The author needs to provide the % increase or decrease rather than just writing ''significantly increased….''.
Response: Thank you very much for your advice. In this study, the agronomic traits were investigated based on previous methods and there were standards for the identification of mutation types. After the agronomic traits of mutant plants were counted, the total number was counted. We only need to consider the number of plants within the scope of the standard identification. Taking plant dwarfing as an example, under the same management conditions, the average plant height of the mutant strain is 20 cm lower than that of the wild type, and it can be identified as dwarfing.
- The discussion is poorly written hence, needs rewriting. The discussion should be further strengthened by adding some more relevant papers. The literature search is insufficient, only few related research papers in the past five years are cited (31, 57%), and add the latest research results appropriately. See the links below if you think it will benefit your discussion.
Response: Thank you very much for your advice. Discussion has been modified in the text. Some new references have been added to our manuscripts.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
The reviewer's comments have been accepted and properly addressed.
