OsChlC1, a Novel Gene Encoding Magnesium-Chelating Enzyme, Affects the Content of Chlorophyll in Rice

Round 1

Reviewer 1 Report

The presented manuscript summarizes experimental data on the novel yellow-green-leaf mutant (ygl9311) obtained a by ⁶⁰Co irradiation. Authors showed that the photosynthetic pigment contents of this mutant were significantly reduced and chloroplast development was retarded compared with that of wild type. On the basis of the genetic analysis, map-based cloning and sequence analysis author suggested a new gene which responsible for the yellow-green leaf phenotype. Mutant using the CRISPR/Cas9 23 system of this gene showed similar phenotype. The obtained experimental data are very nice. However, authors main conclusion that this gene participates on chlorophyll biosynthesis do not fit with the obtained data. No any prove of the chlorophyll biosynthesis products and/or enzymatic evidence of the corresponding gene product for Mg-chelatase activity.

I propose to re-write the presented manuscript without indicating function of the gene product and only discuss the possibility of this gene product for chlorophyll biosynthesis.

Author Response

Dear reviewer，

Thank you very much for your insightful advice. Admittedly, the data we have obtained at present can only show that the gene affects the content of chlorophyll in rice.

Point 1: I propose to re-write the presented manuscript without indicating function of the gene product and only discuss the possibility of this gene product for chlorophyll biosynthesis.

Respones 1: According to your suggestion, we have made the following modifications：

1. We change the title to "OsChlC1, a Novel Gene Encoding Magnesium Chelating Enzyme, Affects the Content of Chlorophyll in Rice" 
2. In the abstract, line 25-26， we rewrite it as “The results indicate that OsChlC1 is the novel gene of Mg-chelatase and affects the content of chlorophyll in Rice”
3. In the discussion, line 407-408, we rewrite it as "In conclusion, OsChlC1 is a newly cloned Mg-chelatase I subunit gene in rice, and its mutation reduces the content of chlorophyll in rice, leading to the phenotype of yellow-green leaves."
4. In the conclusion, line 407-408, we rewrite it as "Therefore, the following conclusion was made: OsChlC1 mutation in the genome of the indica rice cultivar 9311 leads to decrease of chlorophyll content in rice leaves, affects chloroplast development, and results in the phenotype of yellow-green leaves of the mutant ygl9311."

Reviewer 2 Report

Authors submitted manuscript entitled “OsCHS1, a Novel Gene Encoding Magnesium Chelating Enzyme, Regulates Chlorophyll Synthesis in Rice” to Agronomy journal. In this manuscript, authors have identified and functionally characterized a gene in rice which controls chlorophyl synthesis. This work provides good results and manuscript is well written. Some parts of the manuscript need to be improved. I have following suggestions,

It was previously reported that OsCHS1 physically interact with OsF3H, OsF3'H, OsDFR, and OsANS1. Authors have checked the expression of these genes in knockout mutants or not?

Abstract is not very strong. Some sentences are very short and irrelevant.

Authors given the gene name by themselves? There is already a gene CHS, OsCHS1 (LOC_Os11g32650; Os11g0530600) in rice. Can you mention is MSU locus for this gene?

Some papers already mentioned the role of OsCHS1 in rice, so authors should add the enough information about work done on this gene in introduction section.

Information about statistical analyses is missing in methodology.

Insert table 1 as a table, not figure (screenshot).

The manuscript needs to be prepared according to the Agronomy journal format.

Figure 8; phenotypic picture is not very clear.

 

Discussion section needs some sentences about research gaps and possible future studies. 

Author Response

Dear reviewer,

Thank you very much for your insightful advice. According to your suggestion, we revise the manuscript as follows：

Point 1: It was previously reported that OsCHS1 physically interact with OsF3H, OsF3'H, OsDFR, and OsANS1. Authors have checked the expression of these genes in knockout mutants or not?

Respones 1: We are not strict enough about the naming of genes. Thank you again for pointing out our shortcomings. We've changed the name of the gene to “OsChlC1”, meaning Chlorophyll content gene in rice. 

With your reminder, we have noticed that OsCHS is a flavonoid synthesis gene, which was published by Shih et al on Planta in 2008. OsChlC1 and OsCHS1 are two different genes in two unrelated metabolic pathways, so we do not checked the expression of OsF3H, OsF3'H, OsDFR, and OsANS1 in knockout mutants.      

Point 2: Abstract is not very strong. Some sentences are very short and irrelevant.

Respones 2: As the abstract, we have rewritten it according to your suggestion.

Point 3: Authors given the gene name by themselves? There is already a gene CHS, OsCHS1 (LOC_Os11g32650; Os11g0530600) in rice. Can you mention is MSU locus for this gene?

Respones 3: We've changed the name of the gene to “OsChlC1”, meaning Chlorophyll content gene in rice.  

Point 4: Some papers already mentioned the role of OsCHS1 in rice, so authors should add the enough information about work done on this gene in introduction section.

Respones 4: With your reminder, we have noticed that OsCHS is a flavonoid synthesis gene, which was published by Shih et al on Planta in 2008. OsChlC1 and OsCHS1 are two different genes in two unrelated metabolic pathways, so we do not add information related to OsCHS1 in the introduction。

Point 5: Information about statistical analyses is missing in methodology.

Respones 5: We have added chi square test to the genetic analysis method, line 94. In addition, we used the equation of Lichtenthaler when calculating the chlorophyll content in line 115. The analysis method of significant difference (Duncan´s Multiple Range tests) is directly added to the annotation of each figure. Chi square test is used for inheritance pattern analysis of ygl9311 locus in line 200-201

Point 6: Insert table 1 as a table, not figure (screenshot).

Respones 6: We have Inserted table 1 to the manuscript in the form of a table in line 161.

Point 7: The manuscript needs to be prepared according to the Agronomy journal format.

Respones 7: We re-prepared the manuscript on the MDPI template

Point 8: Figure 8; phenotypic picture is not very clear.

Respones 8: We have replaced the previous Figure 8 with a clearer picture in line 333.

Point 9: Discussion section needs some sentences about research gaps and possible future studies. 

Respones 9: As your suggestion, we have added the  research gaps and possible future studies at the end of the discussion between line 411-420.

 

 

Reviewer 3 Report

Major Comments

Line 146-147: How many total SSR markers you have used for mapping and please provide the sequences of their primers as supplementary data file?

Line 178: I shall suggest that confirm or analyze your inheritance pattern results through chi-square test

Line 208-212: I shall suggest that please check the effect of mutation on the activity of other ROS and anti-oxidant species in mutant and compare it with WT.

Although you have knocked out and down-regulated the expression of the gene using CRISPR and RNAi techniques, I shall suggest complementation assay to validate its function.

Line 258: Transcriptome results are not presented, I shall suggest that please provide those results and particularly the data of those 217 differentially expressed genes from which you finalized two genes for further analysis.

Figure 2 does not showing any significant results. Are you sure plant height, no. of productive tillers and no. of spikelet per panicle were non-significant?

Figure 3 and 4: No error bars and significance test results

Figure 5 is not of good quality. Not labeled properly. Results are not clear.

Fig 8 quality is not that good. It should show the results of parameters that can be taken at seedling stage related to the plant phenotype.

 

Minor comments:

Line 56: it should be “… comprises of two parts: …”

Line 86-87: Please remove “To study the genetic pattern of the yellow leaf mutant” from the start of the sentence or end of the sentence.

Line 130: Remove comma (,) after “each containing”.

Line 147: The number of marker you have mentioned in the text is contradicting with the number of markers present in table 1. Please check.

Line 152: Please mention the date when you access the website. Consider this comment for all the links throughout the MS.

Table 1: Which primer was used as forward primer and which one as reverse, not clear. Please check

Line 251: “7” should be written in words rather in digits.

Fig 1, 2 and 3 can be merged.

Fig 8 quality is not that good. It should show the results of parameters that can be taken at seedling stage related to the plant phenotype.

Discussion section needs little improvements technically.

Author Response

Dear reviewer,

Thank you very much for your insightful comments. According to your suggestion, we revise the manuscript as follows：

Point 1: Line 146-147: How many total SSR markers you have used for mapping and please provide the sequences of their primers as supplementary data file?

Response 1: We used seven SSR markers to map the ygl9311 locus. There is  a  clerical error in the first manuscript. We have corrected it in line 153. In order to facilitate people to get the primer sequence conveniently, we have put them in Table 1, line 161 

Point 2: Line 178: I shall suggest that confirm or analyze your inheritance pattern results through chi-square test

Response 2: Chi square test has been added for inheritance pattern analysis of ygl9311 locus in line 200-201

Point 3: Line 208-212: I shall suggest that please check the effect of mutation on the activity of other ROS and anti-oxidant species in mutant and compare it with WT.

Response 3: It is a good suggest that check the effect of mutation on the activity of other ROS and anti-oxidant species in mutant and compare it with WT. Unfortunately, we don't have enough time to complete it in this manuscript. We will carry out this work in the follow-up study. 

Point 4: Although you have knocked out and down-regulated the expression of the gene using CRISPR and RNAi techniques, I shall suggest complementation assay to validate its function.

Response 4: Complementary  assay can really make our results more complete. We have carried out some relevant work, but have not yet obtained data. We have added corresponding instructions at the end of the discussion in line 401-413.

Point 5: Line 258: Transcriptome results are not presented, I shall suggest that please provide those results and particularly the data of those 217 differentially expressed genes from which you finalized two genes for further analysis.

Response 5: I am sorry to think that transcriptome sequencing is a means of mapping ygl9311 locus in this study, so we do not intend to put the details of transcriptome into the manuscript. However, we are willing to submit the data of 217 differentially expressed genes to the reviewer. Please check it in the attachment of the letter.

Point 6: Figure 2 does not showing any significant results. Are you sure plant height, no. of productive tillers and no. of spikelet per panicle were non-significant?

Response 6: With your suggestion, we have added significance analysis to Figure 2. According to the significance analysis, plant height, no. of productive tillers and no. of spikelet per panicle were significant in line 211-212, figture 2.

Point 7: Figure 3 and 4: No error bars and significance test results

Response 7: With your suggestion, we have added bar and significance analysis to Figure 3 and 4 in line 311-316.

Point 8: Figure 5 is not of good quality. Not labeled properly. Results are not clear.

Response 8: Thanks for your reminding, we have submitted a clearer Figure 5 and corrected the label in line 319-323.

Point 9: Fig 8 quality is not that good. It should show the results of parameters that can be taken at seedling stage related to the plant phenotype.

Response 9: We have replaced the previous Figure 8 with a clearer picture in line 333. As you suggested, we would like to put a picture of the phenotype at seed setting stage, but the mutant plants are still in the seedling stage.

Point 10: Line 56: it should be “… comprises of two parts: …”

Response 10: Thank you for your reminder. We have corrected it with "Chl synthesis comprises of two parts" in line 58.

Point 11: Line 86-87: Please remove “To study the genetic pattern of the yellow leaf mutant” from the start of the sentence or end of the sentence.

Response 11: According to your suggestion, we have deleted this sentence “To study the genetic pattern of the yellow leaf mutant”.

Point 12: Line 130: Remove comma (,) after “each containing”.

Response 12: We have removed comma (,) after “each containing”.

Point 13: Line 147: The number of marker you have mentioned in the text is contradicting with the number of markers present in table 1. Please check.

Response 13: We have corrected the number of SSR markers to seven in line 152, which is consistent with Table 1

Point 14: Line 152: Please mention the date when you access the website. Consider this comment for all the links throughout the MS.

Response 14: We have added  website information about SSR marker in line 154.

Point 15: Table 1: Which primer was used as forward primer and which one as reverse, not clear. Please check

Response 15: We have marked forward primer and reverse primer in Table 1 in line 161.

Point 16: Line 251: “7” should be written in words rather in digits.

Response 16: We've replaced "seven" with "7".

Point 17: Fig 8 quality is not that good. It should show the results of parameters that can be taken at seedling stage related to the plant phenotype.

Response 17: We have replaced the previous Figure 8 with a clearer picture in line 333. As you suggested, we would like to put a picture of the phenotype at seed setting stage, but the mutant plants are still in the seedling stage.

Point 18: Discussion section needs little improvements technically.

Response 18: As your suggestion, we have added the  research gaps and possible future studies at the end of the discussion between line 411-420.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors need to spell check!

Author Response

Dear reviewer,

Thank you for your suggestions and work.

Point 1: Authors need to spell check!

Respones 1.  We have made following modifications in the manuscript:

1. We added “generated” in line 16.
2. We correct “ygl9311, was isolated, which dispalyed” to "ygl9311, was isolated: it dispalyed" in line 17.
3. We added “the” in line 18.
4. We changed "chloroplasts development were retarded" to "chloroplast development was retarded" in line 19.
5. We removed the comma in line 22.
6. We changed "mutant" to "mutation" in line 23.
7. We changed indica and japonica to italics in line 25-26.
8. We changed "Mg2+" to "Mg²⁺" in line 41.
9. We changed "grayish green" to "grayish-green" in line 44.
10. We deleted "results" in line 80.
11. We changed "three leaves stage" to "three-leaf stage" in line 110.
12. We changed "sequence" to "sequences" in line 161.
13. We adjusted the format of [29] in line 114.
14. We changed "indica" to "indica" in line 190.
15. We changed "It" to "it" in line 190.
16. We changed "affect" to "affects" in line 216.
17. We deleted " when rice plants were" in line 217.
18. We changed "indicated" to "indicated" in line 221.
19. We deleted "between means" in line 309.
20. We changed ygl9311 to italics in line 321.
21. We removed the space between ygl9311 and comma in line 322.
22. We correct "is comprised of 2 exons and 1 intron" to "comprises two exons and one intron" in line 332.
23. We correct "content" to "contents" in line 352.
24. We added “whereas” in line 390.
25. We correct "can not" to "could not" in line 401.
26. We correct "There" to "there" in line 411.
27. We correct "experiment" to "studies" in line 412.
28. We deleted "that need" in line 412.
29. We correct "also" to "already" in line 413.
30. We correct "can be" to "have been" in line 414.
31. We deleted "at present" in line 414.
32. We correct "We" to "we" in line 416.
33. We correct "choroplast" to "chlorophyll" in line 417.
34. We correct "detect" to "determine" in line 417.
35. We correct "the above mutants" to "different mutants" in line 417.
36. We correct "will" to "need to" in line 419.
37. We deleted "in the future study" in line 420.
38. We correct "decrease of " to "a decrease in" in line 427.

Best wishes,

Reviewer 3 Report

Dear authors,

Thank you for revising your MS. Response is quite satisfactory. However, figures quality is still poor. Please improve the quality.

In addition, mutant name should be italicized in Fig 2-4. Please check and revise the figures.

Best wishes,

Author Response

Dear reviewer,

Thank you for your suggestions and work. We revise the manuscript as follows：

Point 1:  figures quality is still poor. Please improve the quality.

Respones 1: Thank you for your suggestion. We optimized the Figure 1,  Figure 5 and  Figure 8 to improve its quality. 

Point 2: mutant name should be italicized in Fig 2-4. Please check and revise the figures.

Respones 2: We corrected the name of the mutant in Figure 2 - Figure 4 to italics in line 306-316.

Best wishes,