Increasing the Activity of Sugarcane Sucrose Phosphate Synthase Enhanced Growth and Grain Yields in Transgenic Indica Rice

Round 1

Reviewer 1 Report

The authors gave a straightforward experiment to understand how overexpression of sucrose-related genes affect the overall growth and development of rice. It is not as novel, but still a good resource for future studies that could have direct impacts to farmers. The biggest concern I have for this study is it did not provide future directions for the current work. I understand this is not part of the objectives for this study, however I would have like to see that there are next steps that will be taken for this study. A few more points I want to make:

1. Did the transgenic lines have other developmental phenotypes that were observed compared to the NT rice? Were the transgenic lines grain maturity became shorter? Were there differences observed in the architecture of the transgenic lines?

2. Were the transgenic lines more susceptible to tolerant to abiotic or biotic stresses?

3. How was the grain quality (head rice, milling rate, texture, taste?) of the transgenic lines compared to NT rice? I believe this will be an important question to ask as most rice research that improve yield and other yield related traits will be used for consumption. Thus, consumer preference will be a factor into how this study will yield important results for the future. 

Author Response

Response to Reviewer 1 Comments

Thank you very much for your valuable comments. These comments helped me a lot to improve the article. The English language (minor spell check) has been checked and revised. The introduction also has been revised to provide sufficient background, including the appropriate references were added. Some methods that not adequate were revised to provide clear explanations. The revised sentences were yellow highlighted in the manuscript. The revisions were submitted with involvement the comments from Reviewer 2.      

Point 1: Did the transgenic lines have other developmental phenotypes that were observed compared to the NT rice? Were the transgenic lines grain maturity became shorter? Were there differences observed in the architecture of the transgenic lines?

Response 1: Thank you for your suggestion, the differences between the transgenic and NT rice have been presented in the manuscript and the other dereferences in developmental phenotypes will be considered to be observed in next experiment.  There are no different on grain maturity of the transgenic lines and NT rice. We had presented the differences on tiller number and plant high (Table 1), but the other differences in the architecture of the transgenic lines will be observed in the next experiment.  

Point 2: Were the transgenic lines more susceptible to tolerant to abiotic or biotic stresses?

Response 2: Thank for your good comment, the transgenic rice has not been examined for abiotic and biotic stresses. Your comment will be considered to examine the tolerances of the transgenic lines on abiotic stress, since sucrose has acquired an important regulatory function in controlling stress tolerant.

Point 3: How was the grain quality (head rice, milling rate, texture, taste?) of the transgenic lines compared to NT rice? I believe this will be an important question to ask as most rice research that improve yield and other yield related traits will be used for consumption. Thus, consumer preference will be a factor into how this study will yield important results for the future.

Response 3: Thank you very much for your excellent suggestion. Regarding to the grain quality such as milling rate, texture, taste have not presented in this manuscript. The characterization of transgenic rice grain quality will be examined on field trial experiment under supervision of biosafety agency. However, we suggest that cooking properties improve as the amylose content was significantly increased in transgenic rice (Table 2).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments:

1, The introduction could be used in any other paper about SoSPS gene research. The authors should improve it so that the readers can catch the point why this work was conducted. What progress? What problem? Especially in indica rice.

2, line 41. Climate change does no matter with this study. By the way, the reference was also cited secondly.

3, line 48-49. “The role of sucrose accumulation in rice leaves is defined as the ratio of sucrose/starch in leaves”. Why not apply this index in this analysis?

4, line 67. Delete “japonica”, since it is subspecies.

5, line 99-103. In my opinion, the generation should be “T0 seed---T1 plant---T1 seed---T2 plant---T2 seed---T3 plant”.

6, line 103. I’m not sure that “triplicates” here means three biological replicates? The author should state how many individuals per replicate. As mentioned “three independent plants (line 261)”, is it denotes one individual in each block or three individuals in one block?

7, it was confused why SDS, CTAB and sodium lauryl sarcosine were used in the same time. If the young rice leaves were sampled for PCR, any one was ok.

8, line118. “Anur et al., 2020” was cited in wrong way. The primers of npt II gene marker and PCR reaction program should be described in details.

9, line 149. “Sucrose synthase” can be deleted because SuSy was appeared already in line 62. The same case in line 271

10. line182. Starch consist of amylose and amylopectin. It may be incomplete that only amylose starch was analyzed.

11, line 231 and line 25. When the foreign SoSPS1 gene was integrated into rice genome, it should be heterozygous but not homozygous.

12, Fig 2. B and C were upside down.

13, Fig 2., Because SoSPS1 is a foreign gene, NT rice should be no expression of SoSPS1. Or, is it may be the expression of OsSPS genes? Does SoSPS1 share high similarity with six rice SPS genes? How to understand the visible expression of cDNA in 2A and no expression of protein in 2C?

14, Fig 2 and Fig 3. The comma should be replaced by point in the Y coordinate value. e.g. 3.00, 2.50 and so on.

15, Fig 2 and Fig 3, difference in some groups may reach the significance level of 0.01. if so, ** should be indicated.

16, lines 262, 288, 307, 333. The description of significant differences should be uniform

17, line 281. “sucrose is mainly degraded by SAI and SuSy in the leaves and is partly exported for starch synthesis in the seed”, this conclusion was queried to me.

18, Table 2. It seemed that the tiller numbers were much larger than that in field. Would you please add description of the growth condition in green house?

19, Table 2. WT changed to NT.

20, Fig 4. Does heading date segregate in five transgenic lines? If there’s any other trait generated significantly different from NT rice?

21, Fig S2. The name should be changed to SPS#.

Author Response

Response to Reviewer 2 Comments

Thank you very much for your valuable comments and the comments improved the article more clearly. As it was suggested by Reviewer 1, the English language has been checked and revised.  The cited references also have been revised and improved in the manuscript. We think that the research design was appropriate, although description of greenhouse (phytotron) conditions were added and the PCR method was little modified in the material and methods according to Reviewer 2 comments. The results were also improved according to the Reviewer 2 comments, and the conclusions are supported by the results. 

Point 1: The introduction could be used in any other paper about SoSPS gene research. The authors should improve it so that the readers can catch the point why this work was conducted. What progress? What problem? Especially in indica rice.

Response 1: Thank you for your valuable comments and we improved the introduction by adding other papers about SPS gene (line 66 - 69). There is no report about overexpression of SPS gene in indica rice, but only one report in japonica rice (Ono et al, 1999). We found almost no problem with genetic transformation in indica rice, except that to get stable transgenic lines the screening was conducted by PCR analysis, antibiotic kanamycin selection could not be conducted (line 112 - 130).

Point 2: line 41. Climate change does no matter with this study. By the way, the reference was also cited secondly.

Response 2: Thank you very much. To address your comment, we have revised as your suggestion and changed the reference 1, 2 accordingly (line 40 - 44).

Point 3: line 48-49. “The role of sucrose accumulation in rice leaves is defined as the ratio of sucrose/starch in leaves”. Why not apply this index in this analysis?

Response 3: We did not used the ratio sucrose/starch index in our experiment, but directly measured sucrose content using HPLC analysis. Therefore, the sentence “The role of sucrose accumulation in rice leaves is defined as the ratio of sucrose/starch in leaves” was deleted from the manuscript. Although the sentence was eliminated, our result still provide information regarding carbon partitioning from source organ to sink organ.

Point 4: line 67. Delete “japonica”, since it is subspecies.

Response 4: Thank you for your suggestion, the “japonica” is deleted.  

Point 5:  line 99-103. In my opinion, the generation should be “T0 seed---T1 plant---T1 seed---T2 plant---T2 seed---T3 plant”.

Response 5: Thanks, the generation of T1, T2, and T3 were mentioned according to the reference of Xu et al, 2015 [26]. Therefore, we have added the reference at the end of the paragraph to define (line 108 - 109).

Point 6: line 103. I’m not sure that “triplicates” here means three biological replicates? The author should state how many individuals per replicate. As mentioned “three independent plants (line 261)”, is it denotes one individual in each block or three individuals in one block?

Response 6: Thank you. Yes, we are using three biological replicates, means three plants in one replicate, and not individual plant in a replicate. Therefore, we changed “for three independent plants (line 272)” to from three individual plant.

Point 7: , it was confused why SDS, CTAB and sodium lauryl sarcosine were used in the same time. If the young rice leaves were sampled for PCR, any one was ok.

Response 7: Thank you very much for your valuable correction. We used only SDS in the buffer for genomic extraction. Therefore, the CTAB and sodium lauryl sarcosine are removed from the sentence (line 115). We apologized to have mistypes in the material and methods, because in the preliminary analysis mixture of the three chemicals was used and not succeed, replaced by only SDS as described in the reference of Apriasti et al, 2018.    

Point 8: line118. “Anur et al., 2020” was cited in wrong way. The primers of npt II gene marker and PCR reaction program should be described in details.

Response 8: Thank you very much for your correction. We replaced “Anur et al, 2020” with [22]. The reaction program is completed with reaction condition, including the nucleotides sequence of the primer (line 123).

Point 9: line 149. “Sucrose synthase” can be deleted because SuSy was appeared already in line 62. The same case in line 271.

Response 9: Thank you very much. Yes, the sentence “Sucrose synthase” was deleted and replaced by SuSy in line 159 as well as line 283 both for SuSy and SAI.

Point 10: line182. Starch consist of amylose and amylopectin. It may be incomplete that only amylose starch was analyzed.

Response 10: Thank you very much. Yes, you are true that starch consist of amylose and amylopectin, but we found difficulties to measure amylopectin. Therefore, we only used amylose as a representative of starch content.

Point 11: line 231 and line 25. When the foreign SoSPS1 gene was integrated into rice genome, it should be heterozygous but not homozygous.

Response 11: Thank you. Homozygous in the manuscript means that the SoSPS1 gene has been segregated from T1 up to T3 generation according to the Mendelian, and after T3 generation we got stable insertion of SoSPS1 gene or stable transgenic lines and we called homozygous.  

Point 12: Fig 2. B and C were upside down.

Response 12: Thank you for your correction. The Fig 2, B and C were revised by reversing C to B. 

Point 13: Fig 2., Because SoSPS1 is a foreign gene, NT rice should be no expression of SoSPS1. Or, is it may be the expression of OsSPS genes? Does SoSPS1 share high similarity with six rice SPS genes? How to understand the visible expression of cDNA in 2A and no expression of protein in 2C?

Response 13: Thank you for your suggestion. The nucleotides sequence of the primers was designed at the conserve region of SPS gene. Thus, the primers share a high similarity or react with both SoSPS1 and OsSPS gene from rice. Therefore, we changed SoSPS1 gene was replaced by SPS gene in the figure caption. However, the antibody of SPS used in this study was prepared using recombinant protein as the antigen that containing only C-terminal domain of SoSPS1 in order to have a high specificity of the antibody.

Point 14: Fig 2 and Fig 3. The comma should be replaced by point in the Y coordinate value. e.g. 3.00, 2.50 and so on.

Response 14: Thank you for your detail correction. We apologized to not pay attention for the marks (writing number) since we use comma instead of point in our language. Therefore, we replaced comma in the value by point, and simplify the value by omitting 0 in the end of value in the Fig 2 and Fig 3, including replace of comma instead of point in Table 1 and Table 2.

Point 15: Fig 2 and Fig 3, difference in some groups may reach the significance level of 0.01. if so, ** should be indicated.

Response 15: Thank you very much. Yes, we have revised by adding two starts (**) in the Fig 2 and 3 for the significant difference at 0.01, including Table 1 and 2. Therefore, the captions were also revised accordingly.  

Point 16: lines 262, 288, 307, 333. The description of significant differences should be uniform

Response 16: For the uniform of significant differences, we revised in line 318 (Table 1) and 345 (Table 2) by star (*) or (**)

Point 17: line 281. “sucrose is mainly degraded by SAI and SuSy in the leaves and is partly exported for starch synthesis in the seed”, this conclusion was queried to me.

Response 17: Thank you very much. The sentence means that partly sucrose that not been degraded by SAI and SuSy is exported for starch synthesis in the seed. Therefore, we revised the sentence (line 292 - 294).

Point 18: Table 2. It seemed that the tiller numbers were much larger than that in field. Would you please add description of the growth condition in green house?

Response 18: Thank you for your valuable suggestion. Compared to rice planted in the field, the tiller numbers were larger in our experiment, especially on the last observation at three months after plantation (3 MAP). At present time, we do not know the reason, since the rice was cultivated in the green house (phytotron). Therefore, we add the description of the growth condition in the Material and Methods (line 101 - 102). The temperature was adjusted at 25-27 degree Celsius and the humidity at 70-80 %.

Point 19: Table 2. WT changed to NT

Response 19: Thank you, we changed WT to NT in the Table 2.

Point 20: Fig 4. Does heading date segregate in five transgenic lines? If there’s any other trait generated significantly different from NT rice?

Response 20: The heading date was same between transgenic lines and NT that can been seen in Fig 4. At present time, we observed the characters of transgenic lines and NT rice as described in the manuscript. However, we will have more detail observation in the next experiment.  

Point 21: Fig S2. The name should be changed to SPS#.

Response 21: Thank you, we changed to SPS# for T#.

Author Response File: Author Response.pdf