Soybean LEAFY COTYLEDON 1: A Key Target for Genetic Enhancement of Oil Biosynthesis
, Khulood Fahad Alabbosh 3, Abeer Al-Andal 4, Waqas Ahmad 5, Khalid Ali Khan 6 and Jian Zhao 2,7,*Round 1
Reviewer 1 Report
This research explores the functional role of GmLEC1 in soybean seed development and lipid storage reserve accumulation. The GmLEC1 can partially rescue the Arabidopsis leaf and seed's development defects indicating the conserved role of AtLEC1 and GmLEC1. Overexpression of GmLEC1 in both Arabidopsis and soybeans increased the fatty acid content and altered the fatty acid content and composition. The research provides insights into its potential applications for enhancing oil production in soybean crops. I can see the value of the research; however, the English writing is poor. I suggest the authors improve the writing to make the expression clearer.
Some suggestions:
Line 112: It is inappropriate to use 'Usually' at the start of a sentence, and it is very hard to understand the whole sentence.
Line 117: The dark color of atlec1 is relative, the verb is missing
Line 129: figure legend 2B: 'not healthy'- the words are vague
Line 135: GC, the first time shown in the paper, please provide full name
Line 159: 'transformation rate' might be written as 'efficiency'
The semi-quantitative RT-PCR and qRT-PCR results to confirm the transformation of GmLEC1 in soybean hairy roots are shown in Figure 5A, please refer to the figure in result 2.4 when the authors describe the corresponding results (Line 163)
Extensive editing of the English language required
Author Response
Reviewer #1
This research explores the functional role of GmLEC1 in soybean seed development and lipid storage reserve accumulation. The GmLEC1 can partially rescue the Arabidopsis leaf and seed's development defects indicating the conserved role of AtLEC1 and GmLEC1. Overexpression of GmLEC1 in both Arabidopsis and soybeans increased the fatty acid content and altered the fatty acid content and composition. The research provides insights into its potential applications for enhancing oil production in soybean crops. I can see the value of the research; however, the English writing is poor. I suggest the authors improve the writing to make the expression clearer.
Response: We are thankful for your valuable comments, suggestions, and detailed evaluation of our manuscript. Your comments and suggestions were constructive and helped us improve the quality of our manuscript. We have carefully addressed all queries and revised the manuscript in light of your valuable comments and suggestions. The changed parts are highlighted in blue in the revised manuscript. The detailed response to reviewers’ comments is given below. We hope that the responses to your questions and subsequent amendments in the revised manuscript are satisfactory to recommend its acceptance for publication.
Some suggestions:
Comment #1
Line 112: It is inappropriate to use 'Usually' at the start of a sentence, and it is very hard to understand the whole sentence.
Response: Thank you for the correction. The sentence was corrected at line 138 of the revised manuscript
Comment #2
Line 117: The dark color of atlec1 is relative, the verb is missing.
Response: Thank you for the correction. Correct verb was added at the line 142 in the revised manuscript
Comment #3
Line 129: figure legend 2B: 'not healthy'- the words are vague.
Response: Thank you for the correction. Correction was made at the line 152 (legend of Fig.2) of the revised manuscript
Comment #4
Line 135: GC, the first time shown in the paper, please provide full name
Response: Thank you for the correction. Full form of the abbreviation was provided at the line 353 of the revised manuscript
Comment #5
Line 159: 'transformation rate' might be written as 'efficiency'.
Response: Thank you for the correction. The word ‘Transformation rate’ was replaced with ‘high efficiency’ at the line 185 of the revised manuscript
Comment #6
The semi-quantitative RT-PCR and qRT-PCR results to confirm the transformation of GmLEC1 in soybean hairy roots are shown in Figure 5A, please refer to the figure in result 2.4 when the authors describe the corresponding results (Line 163)
Response: Thank you for the correction. An additional supplementary figure 3 was provided that shows qRT results of five representative transgenic soybean hairy root lines and figure was referred at the line 188 of the revised manuscript.
Reviewer 2 Report
In this manuscript, the authors investigated the role of the Glycine max transcription factor LEAFY COTYLEDON 1 (LEC1) in seed development and other tissues monitoring Arabidopsis and soybean transgenic plants. They observed that LEC1 overexpression significantly increased the amount of triacylglycerol and changed the fatty acid composition in Arabidopsis seeds and in soybean hair roots. In addition, they found a decrease in starch accumulation in seeds as well as changes in cotyledon phenotype. With these data, the authors concluded that GmLEC1 controls seed development and regulates seed storage compounds. In general, the manuscript has some points that need to be improved.
- The English needs to be extensively revised. Some sentences follow this example below, in which the authors use two verbs at different times to control the sentence.
“In addition, it induces embryonic characteristics in true leaves, which lack trichomes and increased level of stored lipids was found in overexpressed seeds”
Several other sentences have similar problems. Also, pay attention to typing errors
- Line 20 “…overexpressed seeds and vegetative tissues”. Genes are overexpressed, not tissues.
- Lines 40 and 41. … glycolysis, chain elongation, condensation, and desaturation of glycerolipid biosynthesis are not key reactions; instead, they are metabolic pathways.
- Line 73. The item, “2.1 Identification and expression in soybean plant tissues” needs to be clear that they are denoting gene identification and expression.
- What is the mean of SAM; Figure 1B
- Please, include in the methods the accession numbers of sequences used in Figure 1A
- When describing Figures 3 and 4, please pay attention to include the fatty acid nomenclature used in the figures. For example: indicates in the text that oleic acid is (18:1), palmitic acid is (16:0), eicosonic acid, etc.
- Explain in the legend of Figure 4 what mean St and Gus in relation to GmLEC1.
- With regard to RT-qPCR analyses, the authors say that these data were obtained according to Manan et al., 2017. Consulting this paper, these authors claim that they used a single soybean ACTIN gene as a reference. Thus, have the authors certified that this soybean ACTIN gene has stable expression in the conditions assayed in the present manuscript?
- The item “4.3 Arabidopsis transformation and hairy root induction”; also is relative to soybean transformation, thus it is necessary to inform this.
Extensive editing of English language required
Author Response
Reviewer #2
In this manuscript, the authors investigated the role of the Glycine max transcription factor LEAFY COTYLEDON 1 (LEC1) in seed development and other tissues monitoring Arabidopsis and soybean transgenic plants. They observed that LEC1 overexpression significantly increased the amount of triacylglycerol and changed the fatty acid composition in Arabidopsis seeds and in soybean hair roots. In addition, they found a decrease in starch accumulation in seeds as well as changes in cotyledon phenotype. With these data, the authors concluded that GmLEC1 controls seed development and regulates seed storage compounds. In general, the manuscript has some points that need to be improved.
Response: We are thankful for your valuable comments, suggestions, and detailed evaluation of our manuscript. Your comments and suggestions were constructive and helped us improve the quality of our manuscript. We have carefully addressed all queries and revised the manuscript in light of your valuable comments and suggestions. The changed parts are highlighted in blue in the revised manuscript. The detailed response to reviewers’ comments is given below. We hope that the responses to your questions and subsequent amendments in the revised manuscript are satisfactory to recommend its acceptance for publication.
Comment #1
The English needs to be extensively revised. Some sentences follow this example below, in which the authors use two verbs at different times to control the sentence.
Response: Thank you for the suggestion, whole manuscript was extensively revised for spelling, grammar, and typos errors.
Comment #2
“In addition, it induces embryonic characteristics in true leaves, which lack trichomes and increased level of stored lipids was found in overexpressed seeds”
Response: Thank you for the correction. Sentence was restructured at lines 55-58 of the revised manuscript
Comment #3
Several other sentences have similar problems. Also, pay attention to typing errors
Response: Thank you for the suggestion, whole manuscript was extensively revised for spelling, grammar, and typos errors.
Comment #4
Line 20 “…overexpressed seeds and vegetative tissues”. Genes are overexpressed, not tissues.
Response: Thank you for the correction. Sentence was rephrased to express its actual meaning at lines 26-27 of the revised manuscript
Comment #5
Lines 40 and 41. … glycolysis, chain elongation, condensation, and desaturation of glycerolipid biosynthesis are not key reactions; instead, they are metabolic pathways.
Response: Thank you for the correction. The word ‘key metabolic pathways’ was added at line 51 of the revised manuscript.
Comment #6
Line 73. The item, “2.1 Identification and expression in soybean plant tissues” needs to be clear that they are denoting gene identification and expression.
Response: Thank you for the correction. The word ‘Gene’ was added within the heading of section 2.1 (line 99) of the revised manuscript.
Comment #7
What is the mean of SAM; Figure 1B
Response: SAM represents the ‘shoot apical meristem’. It was mentioned in the legend of figure 1B (line 117) of the revised manuscript.
Comment #8
Please, include in the methods the accession numbers of sequences used in Figure 1A
Response: Thank you for the suggestion, accession number of the proteins used in figure 1A were provided in the supporting data file under “Supplementary Data 1” of the revised manuscript.
Comment #9
When describing Figures 3 and 4, please pay attention to include the fatty acid nomenclature used in the figures. For example: indicates in the text that oleic acid is (18:1), palmitic acid is (16:0), eicosonic acid, etc.
Response: Thanks for the suggestion. Fatty acid nomenclature was described in the legend of figure 3 (lines 178-179) and mistakes were corrected throughout the whole manuscript.
Comment #10
Explain in the legend of Figure 4 what mean St and Gus in relation to GmLEC1.
Response: Thank you for the correction. ‘St’ represents ‘standard/positive control’, GUS represents ‘Control’ relative to GmLEC1. The terms are explained in the legend of Figure 4 (line 205-206).
Comment #11
With regard to RT-qPCR analyses, the authors say that these data were obtained according to Manan et al., 2017. Consulting this paper, these authors claim that they used a single soybean ACTIN gene as a reference. Thus, have the authors certified that this soybean ACTIN gene has stable expression in the conditions assayed in the present manuscript?
Response: Soybean plants used in the current study were grown under the same conditions as described previously in Manan et al., 2017. Hence, authors assume that this soybean ACTIN gene has stable expression in the conditions assayed in the present manuscript
Comment #12
The item “4.3 Arabidopsis transformation and hairy root induction”; also is relative to soybean transformation, thus it is necessary to inform this.
Response: The heading of the section 4.3 was modified at line 340 of the revised manuscript that better reflects the contents of the section.
Reviewer 3 Report
The presented work is devoted to studying the way to increase oil accumulation in soybean seeds using genetic transformation. The work has been done extensively and interestingly, the purpose of the work is relevant. To improve the perception of the work, I ask the authors to still specify in Figure 1 what S1 and S6. All captions must be reviewed. Overall the work makes a good impression.
Comments for author File: 
Comments.pdf
English needs some correction
Author Response
Reviewer #3
The presented work is devoted to studying the way to increase oil accumulation in soybean seeds using genetic transformation. The work has been done extensively and interestingly, the purpose of the work is relevant. The article represents «Soybean LEAFY COTYLEDON 1: A Key Target for Genetic Enhancement of Oil Biosynthesis» a complete and relevant study. The transcription factor LEAFY COTYLEDON 1 is important in the regulation of embryonic development and seed maturation. Fundamentally shown to be involved GmLEC1in the regulation of fat metabolism in plants. Overall the work makes a good impression.
Response: We are thankful for your valuable comments, suggestions, and detailed evaluation of our manuscript. Your comments and suggestions were constructive and helped us improve the quality of our manuscript. We have carefully addressed all queries and revised the manuscript in light of your valuable comments and suggestions. The changed parts are highlighted in blue in the revised manuscript. The detailed response to reviewers’ comments is given below. We hope that the responses to your questions and subsequent amendments in the revised manuscript are satisfactory to recommend its acceptance for publication.
Comment #1
To improve the perception of the work, I ask the authors to still specify in Figure 1 what S1 and S6. All captions must be reviewed.
Response: Thanks for the suggestion. The S1- S6 were described in the material section at lines 333-337 of the revised manuscript.
Comment #2
In the Abstract part, clarify the name of the trichome, line 22? this is not entirely clear.
Response: Thanks for the suggestion. Correction is made in the abstract.
Comment #3
For a better understanding, it would be better to separate those experiments where Arabidopsis transgenes were used and separately hairy roots. Since the results obtained using Arabidopsis mutants were extrapolated to the hairy roots of soybean.
Response: Result section was improved according to your valuable comments.
Comment #4
In the Introduction, the authors quite sufficiently described the current state of the problem. Line 53 - specify the crop, cabbage is rapeseed - "in other oil crops like maize and brassica». I think lines 67-70 should be removed, since this already relates to the results.
Response: Thanks for the suggestion. Lines 67-70 were removed from the revised manuscript.
Comment #5
In part of the Results, to improve the understanding of the material, it is necessary to clarify where which objects are - Arabidopsis and soybean.
Response: Thanks for the suggestion. Methods and result sections were revised to clarify the ambiguities.
Comment #6
Please look at the captions, especially Figure 5. Add confidence and correct errors of the mean. Figure 4 is close to the understandable version.
Response: Thanks for the correction. Figure captions were modified and details were provided accordingly.
Comment #7
Please, in the Methods, specify which gene encodes what and give its sequence (5’-3’, 3’-5’).
Response: Coding DNA Sequences (CDS) were provided as supporting information in ‘supplementary data 2’.
Comment #8
In the Discussion part, it is necessary to connect the literature data with the results and give figure numbers where necessary and logical. This was not done by the authors to the fullest extent, since there are no references to the Figures.
Response: Thank you for valuable suggestion. In the discussion section, results were discussed in the light of most relevant literature and finding of the current study with figure reference.
Comment #9
The Abstract and Conclusion read- «Main downstream target of LEC1 are GL2 and WRI1, which participates in fatty acid biosynthesis and trichome formation by regulating phytohormones». But there is no data about phytohormones in the work and they were not determined by the authors.
Response: Thank you for raising an important point. As, the plant development and synthesis of various storages compounds are regulated by a complex network of regulators which include transcription factors, phytohormones and it also depends on the environmental conditions. The impact of phytohormones and their relation with master regulators is part of our ongoing project which we will present in the future publications.
Comment #10
There is only the data presented in Figure 5. Please, make it clear.
Response: Thanks for the suggestion. Figure 5 shows the impact of GmLEC1 upregulation on key genes of various interconnected metabolic pathways
Comment #11
Сonfirm, please
Phyozome/ phytozome -?
Eicosonic acid- line 147
Response: Thanks for the correction.
Comment #12
Please review this and make the necessary materials and corrections.
Response: Manuscript was revised according to your valuable suggestion.
Reviewer 4 Report
The manuscript entitled “Soybean LEAFY COTYLEDON 1: A Key Target for Genetic Enhancement of Oil Biosynthesis” reviewed thoroughly. The manuscript has significant scope in investigating oil biosynthesis in crop plants. The authors have made efforts to delineate the role of GmLEC1 in seed development and compositions related to lipids. The manuscript is in good form for accepting for publication. There are only few minor suggestions to authors for making corrections before acceptance.
All the abbreviations should be expanded at their appearance in the manuscript.
Line number 54 in the introduction, check the spelling ‘stud’, it should be ‘studies’
It is suggested to reorient the study results more toward lipid profiles than other compositions and seed shapes as the article focuses on oil biosynthesis.
The discussion section demands attention as the authors fail to discuss the extremely good results in the dimensions of practical utility. The knowledge generated holds potential scope in oil seed breeding and improvement. Hence, a comprehensive discussion should be presented.
The manuscript may be accepted for publication after minor review.
The English language must be read and edited by a native English speaker to improve the language quality of the manuscript.
Author Response
Reviewer #4
The manuscript entitled “Soybean LEAFY COTYLEDON 1: A Key Target for Genetic Enhancement of Oil Biosynthesis” reviewed thoroughly. The manuscript has significant scope in investigating oil biosynthesis in crop plants. The authors have made efforts to delineate the role of GmLEC1 in seed development and compositions related to lipids. The manuscript is in good form for accepting for publication. There are only few minor suggestions to authors for making corrections before acceptance. The manuscript may be accepted for publication after minor review.
Response: We are thankful for your valuable comments, suggestions, and detailed evaluation of our manuscript. Your comments and suggestions were constructive and helped us improve the quality of our manuscript. We have carefully addressed all queries and revised the manuscript in light of your valuable comments and suggestions. The changed parts are highlighted in blue in the revised manuscript. The detailed response to reviewers’ comments is given below. We hope that the responses to your questions and subsequent amendments in the revised manuscript are satisfactory to recommend its acceptance for publication.
Comment #1
All the abbreviations should be expanded at their appearance in the manuscript.
Response: Thank you for the suggestion. Full form of all abbreviations was provided at lines 40, 45, 46, 73, 74, 222 and 223 of the revised manuscripts.
Comment #2
Line number 54 in the introduction, check the spelling ‘stud’, it should be ‘studies’
Response: Thank you for the correction.
Comment #3
It is suggested to reorient the study results more toward lipid profiles than other compositions and seed shapes as the article focuses on oil biosynthesis.
Response: Thank you for the suggestion. The results and discussion were modified according to your valuable suggestions.
Comment #4
The discussion section demands attention as the authors fail to discuss the extremely good results in the dimensions of practical utility. The knowledge generated holds potential scope in oil seed breeding and improvement. Hence, a comprehensive discussion should be presented.
Response: Thank you for the suggestion. The discussion was modified according to your valuable suggestions.
