CRISPR/Cas9-Mediated Targeted Mutagenesis of GmEOD1 Enhances Seed Size of Soybean

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Minor spelling check is required

Author Response

Responses to reviewer 1：

We sincerely appreciate your valuable time and effort in reviewing our manuscript titled "CRISPR/Cas9-mediated Targeted Mutagenesis of GmEOD1 Enhances Seed Size of Soybean." We are pleased to receive your positive feedback on the quality of the writing and the overall theme of our research. Your comments provide us with valuable insights for improving the manuscript, and we would like to address each of your suggestions in detail.

1. Language and Repetition:

Responses: Thank you for pointing out the need for careful language editing.  We thoroughly revised the text to ensure that redundant phrases are removed and the overall language quality is enhanced.

2. Missing Conclusion:

Responses: We added a comprehensive conclusion that effectively summarizes the key findings of our study and highlights their significance. The conclusion part is as followings: "Conclusion: In conclusion, our study has successfully demonstrated the efficacy of CRISPR/Cas9-mediated targeted mutagenesis of GmEOD1 in enhancing soybean seed size. The resulting mutant displayed larger seeds and increased 100-seed weight without compromising plant productivity. This holds promise for improving yield and nutritional content in soybeans. The exploration of GmEOD1 sequence variations among different cultivars establishes a link between specific haplotypes and higher 100-seed weight, indicating the potential for targeted breeding efforts. Our transcriptomic analysis highlighted distinct gene expression patterns during seed development, revealing stages dedicated to growth, carbon accumulation, and nutrient storage. The identification of key regulatory genes through WGCNA provides valuable insights into the complex genetic regulation of seed size. Overall, this study provides essential groundwork and valuable resources for enhancing soybean seed size through molecular breeding strategies, offering opportunities for sustainable agricultural improvements and better quality soybean production." Please check the Line 545-558.

3. Discussion and References:

Responses: We appreciate your suggestion to incorporate new and relevant references in the discussion section. We diligently search for recent references that contribute to the depth and breadth of the discussion. Furthermore, we will ensure that references older than 10 years are only cited if absolutely necessary for providing historical context or foundational knowledge. Please check the reference 23-29.

Once again, we extend our gratitude for your thorough evaluation of our manuscript. Your feedback greatly aids in improving the quality and impact of our work. We are committed to addressing all your concerns and making the necessary revisions to deliver a manuscript that meets the highest standards.

Author Response File: Author Response.docx

Reviewer 2 Report

In the manuscript by Han Yu, Juan Zhao, Tingting Wu, Bingjun Jiang, Cailong Xu, Yupeng Cai, Jialing Dong, Tianfu Han, Li Chen, Shan Yuan, Shi Sun “CRISPR/Cas9-mediated targeted mutagenesis of GmEOD1 enhances seed size of soybean” the characteristics of the mutants with targeted mutagenesis of the gene of E3 ubiquitin ligase GmEOD1 in the soybean cultivar Jack are presented. The findings are of interest for agriculture, since the resulting eod1 mutants possessed the significant increase in seed size without impairing the physiological seed quality with the increased the nutritional value.

This study may be published after correcting a number of inaccuracies.

- L. 30-32: The last sentence of the Abstract “This study provides a valuable theoretical basis and excellent germplasm resources for soybean seed size molecular breeding, facilitating easy selection for seed traits to improve soybean quality” as well as the sentence in the L. 86-88 “These findings provide a vital theoretical foundation and superior germplasm resources for the subsequent molecular breeding of seed size in soybean and other crops” are unclear.   Did you mean that “This study provides a valuable theoretical basis and excellent opportunities for genetic editing of germplasm cells with subsequent molecular soybean seed size breeding, facilitating easier seed selection to improve soybean quality”? Please, check and modify these sentences in such a way as to convey the relevant information to the reader.

- L. 65-67: If DA1 is a ubiquitin protein receptor, please decipher the abbreviation “DAR1”, since you use both of them in the text.

- L. 67-76: After the sentences “EOD1/BB mutants show an increase in seed size, indicating that EOD1/BB may also negatively regulate seed and organ size alone” the reference to the literature data is missed; “Further studies show that both DA2 and EOD1/BB can ubiquitinate DA1 at multiple sites, thereby activating the peptidase activity of DA1, which can lyse various growth regulators to regulate the growth of seeds and organs” the reference to the literature data is missed; “In addition, DA2 and EOD1/BB are also regulated by DA1 feedback” the reference to the literature data is missed; “The plant-specific negative regulator of APC, SAMBA, can regulate seed size by controlling cell proliferation” the reference to the literature data is missed; “Further studies found that Samba could cooperatively enhance the seed size of da1-1 and eod1-2, indicating that it may have functional redundancy or interaction with DA1 and EOD1/BB in influencing seed size” the reference to the literature data is missed.

L. 91. Please, give the Latin name of the research object (soybean cultivar Jack).

- L. 210-211: Didn’t you use the elongation stage?

- L. 252: “got no” doesn't sound English

- L. 258-279: This section contains a description of the data in Figure 3, but there is no letter numbering of individual figures in Figure 3. This complicates the perception of the results. At the same time, letter designations are present in the lefend to this Figure. Please correct. L. 270-273 of this section: “The results (Figure 3) showed that the soybean glycoside content of the mutant seeds was lower than that of the WT (p < 0.05) while there were no significant differences in the total concentrations of soybean xanthin, genistein, soybean xanthin, soybean xanthin glycogen, genistein, and isoflavones (p > 0.05).” Where are the data for glycoside content in the Figure 3? At that, the data for isoflavones in the Figure 3 show that their content was lower in eod1 than in the WT. Please, check the data in the Figure 3.

- The references to Figure S1 and Figure S3 are missed

-The reference to Table S2 is in L.242 that is after the reference to Table S6. Probably, it is worth changing the order of the numbering of Tables in Supplementary section.

- There are no spaces in the legends to the Tables and Figures in Supplementary section. Please, correct.

-The legend to Figure 2 is unclear. “INS1, ACCAAT. INS2, TGGAAGAGAGGGAGA.” What does it mean? This is incomprehensible from the common sense and invalid form of English grammar.

- Figure S3: Please explain in the Figure legend, what is the difference between the five seeds shown in each of the two rows for the mutant and wild type, respectively. Are these seeds of different ages?

- Figure S5 is incorrectly structured, letters intersect with each other. Please correct.

Minor English editing are required according to my comments

Author Response

To reviewer 2：

In the manuscript by Han Yu, Juan Zhao, Tingting Wu, Bingjun Jiang, Cailong Xu, Yupeng Cai, Jialing Dong, Tianfu Han, Li Chen, Shan Yuan, Shi Sun “CRISPR/Cas9-mediated targeted mutagenesis of GmEOD1 enhances seed size of soybean” the characteristics of the mutants with targeted mutagenesis of the gene of E3 ubiquitin ligase GmEOD1 in the soybean cultivar Jack are presented. The findings are of interest for agriculture, since the resulting eod1 mutants possessed the significant increase in seed size without impairing the physiological seed quality with the increased the nutritional value.

This study may be published after correcting a number of inaccuracies.

1. L. 30-32: The last sentence of the Abstract “This study provides a valuable theoretical basis and excellent germplasm resources for soybean seed size molecular breeding, facilitating easy selection for seed traits to improve soybean quality” as well as the sentence in the L. 86-88 “These findings provide a vital theoretical foundation and superior germplasm resources for the subsequent molecular breeding of seed size in soybean and other crops” are unclear.   Did you mean that “This study provides a valuable theoretical basis and excellent opportunities for genetic editing of germplasm cells with subsequent molecular soybean seed size breeding, facilitating easier seed selection to improve soybean quality”? Please, check and modify these sentences in such a way as to convey the relevant information to the reader.

Responses: Thanks for your suggestions. We revised the manuscript as following: L31-33: “This study provides a valuable theoretical basis and excellent opportunities for genetic editing of germplasm cells with subsequent molecular soybean seed size breeding, facilitating easier seed selection to improve soybean quality”. L95-97: These findings provide a vital theoretical foundation and superior genetic editing germplasms for the subsequent molecular seed size breeding in soybean and other crops.

2. L. 65-67: If DA1 is a ubiquitin protein receptor, please decipher the abbreviation “DAR1”, since you use both of them in the text.

Responses: Thanks for your suggestions. We added the abbreviation of DAR1 (DA1-related protein) into the manuscript (Line 74).

3. L. 67-76:After the sentences “EOD1/BB mutants show an increase in seed size, indicating that EOD1/BB may also negatively regulate seed and organ size alone” the reference to the literature data is missed; “Further studies show that both DA2 and EOD1/BB can ubiquitinate DA1 at multiple sites, thereby activating the peptidase activity of DA1, which can lyse various growth regulators to regulate the growth of seeds and organs” the reference to the literature data is missed; “In addition, DA2 and EOD1/BB are also regulated by DA1 feedback” the reference to the literature data is missed; “The plant-specific negative regulator of APC, SAMBA, can regulate seed size by controlling cell proliferation” the reference to the literature data is missed; “Further studies found that Samba could cooperatively enhance the seed size of da1-1 and eod1-2, indicating that it may have functional redundancy or interaction with DA1 and EOD1/BB in influencing seed size” the reference to the literature data is missed.

Responses: Thanks for your kindly reminder. We added the 5 related references, please check the Line 72-84, and the reference 23-29.

4. L.91. Please, give the Latin name of the research object (soybean cultivar Jack).

Responses: Thanks for your good suggestion. We showed the Latin name of soybean at the first appearance in Line 38.

5. L. 210-211: Didn’t you use the elongation stage?

Responses: Thanks for your excellent advice. Since our major purpose is to focus on exploring the expression differences of the key genes that controlling the seed development, then we sampling both the seed and leaves from the beginning to the end of the seed formation. Admittedly, the expression of regulatory genes related to the elongation stage before the seed formation may also affect the seed development and the seed size. We may continue to supplement experiments related to this stage in the coming research. Please check the Line 184-187.

6. L. 252: “got no” doesn't sound English

Responses: Thanks for your advice We revised it into “exhibited no” in Line 266.

7. L. 258-279: This section contains a description of the data in Figure 3, but there is no letter numbering of individual figures in Figure 3. This complicates the perception of the results. At the same time, letter designations are present in the lefend to this Figure. Please correct.L. 270-273 of this section: “The results (Figure 3) showed that the soybean glycoside content of the mutant seeds was lower than that of the WT (p < 0.05) while there were no significant differences in the total concentrations of soybean xanthin, genistein, soybean xanthin, soybean xanthin glycogen, genistein, and isoflavones (p > 0.05).” Where are the data for glycoside content in the Figure 3? At that, the data for isoflavones in the Figure 3 show that their content was lower in eod1 than in the WT. Please, check the data in the Figure 3.

Responses: Thanks for your kindly suggestions. 1)We added the letter numbers of a-c in Figure 3. 2) We retained the academic names for soybean isoflavone components as follows: Daidzein, Daidzin, Genistein, Genistin, Glycitein, Glycitin in Line 283-284. 3) We substituted the “glycoside ” into “Daidzin” in Line 285, Line 463. 4) The total isoflavones content of eod1 mutant (108.79±26.24 μg/mL) was not significantly different with the control (140.71±19.88 μg/mL, p > 0.05) in Figure 3b.

8. The references to Figure S1 and Figure S3 are missed

Responses: Thanks for your kindly reminder. Please check the citations of Figure S1 in Line 120; Figure S3 in Line 187.

9. The reference to Table S2 is in L.242 that is after the reference to Table S6. Probably, it is worth changing the order of the numbering of Tables in Supplementary section.

Responses: Thanks for your kindly reminder. Please check the first citation of Table S2 in Line 140.

10. There are no spaces in the legends to the Tables and Figures in Supplementary section. Please, correct.

Responses: Thanks for your kindly reminder. We have corrected the whole supplementary information section about the accidental deletion of the spaces. Please check the titles of the supplementary tables and figures.

11. The legend to Figure 2 is unclear. “INS1, ACCAAT. INS2, TGGAAGAGAGGGAGA.” What does it mean? This is incomprehensible from the common sense and invalid form of English grammar.

Responses: Thanks for your advises. The INS is the abbreviation of insertion, and we described it more clearly as following: the INS1, the insertion of ACCAAT. INS2, the insertion of TGGAAGAGAGGGAGA. Please the note of the Figure 4.

12. Figure S3: Please explain in the Figure legend, what is the difference between the five seeds shown in each of the two rows for the mutant and wild type, respectively. Are these seeds of different ages?

Responses: Thanks for your suggestions. We added the five stages numbering into the figure. The seeds and leaves of eod1 T₂ generation and WT were sampled at 0 d (stage 1st), 10 d (stage 2nd), 20 (stage 3rd), 30 (stage 4th), 40 (stage 5th) stage since R5 stage R5 stage. Please check the Figure S3.

13. Figure S5 is incorrectly structured, letters intersect with each other. Please correct.

Responses: Thanks for your kindly reminder. We have modified the Figure S5 to avoid the letters overlap.

Author Response File: Author Response.docx

Reviewer 3 Report

A well streamlined site-directed mutagenesis project in order to improve seed size and or grain yield of commercial soybean line. Knock-out mutation of E3 ubiquitin protein ligase EOD1 gene in soybean by crispr/cas9 seem to produce larger seeds and TGW and the T2 homozygous mutant lines is a good starting point for soybean breeding. The introduction is missing a short account of previously identified seed-size induced mutants in soybean and the discussion could eventually follow up by compare these to the site-directed mutations. It is not well described how the new mutant lines were grown for evaluation of seed traits and RNAseq expression analysis. Line 95 "natural condition", but it is seems to be in pots out-doors but not in the soil (field condition). This can strongly affect the RNAseq profile and authors considerations would be needed in this regard. Also include management, irrigation, fertilization, pest management.

What is anticipated yield in terms of kilogram seeds harvested per hectar compared with the parent line? Seed size is improved by there can still be a yield penalty.

What do the authors mean by protein quality? They only measure protein content (and oil content).

Lin 24, 27 "focused" seems to be a wrong term to use in this context.

correct "Aanalyses" occur several times

Author Response

Responses to reviewer 3：

1. A well streamlined site-directed mutagenesis project in order to improve seed size and or grain yield of commercial soybean line. Knock-out mutation of E3 ubiquitin protein ligase EOD1 gene in soybean by crispr/cas9 seem to produce larger seeds and TGW and the T2 homozygous mutant lines is a good starting point for soybean breeding. The introduction is missing a short account of previously identified seed-size induced mutants in soybean and the discussion could eventually follow up by compare these to the site-directed mutations.

Responses: Thanks for your suggestions. We added the brief introduction of the relate molecular breeding pathways as followings: “The essential genes within the ubiquitin-proteasome pathway, MAPK signaling pathway, transcriptional regulatory factors, G protein signaling pathway, IKU pathway, and plant hormone regulation pathway emerge as pivotal focal points for enhancing seed size through breeding. These critical genetic targets have been harnessed to create germplasm resources capable of effecting significant changes in seed size.” Please check the Line 59-64.

2. It is not well described how the new mutant lines were grown for evaluation of seed traits and RNAseq expression analysis. Line 95 "natural condition", but it is seems to be in pots out-doors but not in the soil (field condition). This can strongly affect the RNAseq profile and authors considerations would be needed in this regard. Also include management, irrigation, fertilization, pest management.

Responses: Thanks for your suggestions. Seeds of eod1 mutants and the control were germinated in pots with soil and vermiculite (1:1) in a controlled culture room at 28 ℃ with 50% humidity under about 16h light/8h night conditions and grown under natural conditions in Changchun, China (N43°59’, E125°24’). Regarding irrigation, the plants received regular and appropriate watering on a weekly basis to maintain adequate soil moisture. Fertilization was carried out using suitable fertilizers to meet the nutritional requirements of the plants. In terms of pest management, necessary measures were taken to monitor and control any pests that could potentially affect the health of the plants."

3.What is anticipated yield in terms of kilogram seeds harvested per hectar compared with the parent line? Seed size is improved by there can still be a yield penalty.

Responses: Thanks for your suggestions. Currently, we have only conducted preliminary yield estimations for the mutant individual plants, which showed no significant difference compared to the control. The specific yield effects will need to be assessed through field experiments.

4.What do the authors mean by protein quality? They only measure protein content (and oil content).

Responses: Thanks for your suggestions. We substituted the “protein quality” into “protein content” in Line 273.

5. Line 24, 27 "focused" seems to be a wrong term to use in this context.

Responses: Thanks for your suggestions. We revised into “focussed” in Line 25, Line 27, Line 485, Line 492.

6. correct "Aanalyses" occur several times

Responses: Thanks for your kindly reminders. We corrected into “analysis” in Line 54, Line 159, Line 170, Line 178, Line 195.

Author Response File: Author Response.docx