Comparative Transcriptome Analysis Reveals Different Low-Nitrogen-Responsive Genes in Pepper Cultivars

Round 1

Reviewer 1 Report

The article ”Comparative Transcriptome Analysis Reveals Different Low-nitrogen-responsive Genes in Low-nitrogen-sensitive and Low-nitrogen-tolerant Pepper (Capsicum annum L.) Varieties” by Chunping Wang and coworkers presents a next generation sequencing analysis of the transcriptome of a low-N tolerant and a low-N sensitive pepper variety.

 

In a phenotypic screen of 100 pepper varieties 2 varieties are identified, one of which (750-1) is tolerant and another (ZCFB) which is not tolerant to 4 weeks low-N stress.

 

In a classical RNAseq approach using 3 biological repetitions of pools of 15 plants of each variety the transcriptomes are established and analyzed. Differential analysis identifies about 100-150 DEG that are not related to N metabolism (the remaining 2,000-3,000 DEG are related to N metabolism). The authors discuss a possible role of these DEG in low N tolerance. No functional validation of candidate genes is made, but this is not the intention of the authors. Taken together, the work presents a list of candidates whose further analysis might help to identify genes that could be used to design/breed low N-tolerant pepper plants.

 

Comments:

 

Line 57: “higher” is more adequate than ”increased”.

Line 94: “buds” Do you mean seedlings or buds?

Line 140: one single reference gene is sometimes not sufficient. Did you verify that it does not change expression?

Line 209: Please explain the procedure how you selected the 8 genes randomly.

Line 238/239: I am not sure what you mean exactly by “exclude transcripts expressed at low levels”? Do you mean low changes in expression between the 2 conditions or do you mean low level transcripts (by low number of counts). Please clarify.

Line 340: Autophagy might also be involved in lipid metabolism.

 

Figure 2 SCR6 vs STR8: I do not quite understand how you identified 27,000 no-DEG while at the same time you could only identify about 20,000 genes in total for STR8? Please clarify.

Table 3: Do you have an explanation why there are only about 20,000 genes found in STR8?

Figure 5 and 6: The labeling is not correct for Sxx samples.

Figure 7: You could add gene function to all genes (if known).

Author Response

Dear Professor:

We are very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in manuscript. The responses to your comments are as follows.

1. Line 57: “higher” is more adequate than ”increased”.

Response: Thank you for your perfect advice. The word “increased” has been replaced by “higher”.

 

2. Line 94: “buds” Do you mean seedlings or buds?

Respone: We mean small buds geminating from the seed.

 

3. Line 140: one single reference gene is sometimes not sufficient. Did you verify that it does not change expression?

Response: Thank you for your advice. However, in this paper we didn’t conduct experiment to verify the expression of the reference gene alone. By referring to previous similar studies on pepper and other crops, we choose actin gene (AY572427) as the internal reference gene.

 

4. Line 209: Please explain the procedure how you selected the 8 genes randomly.

Response: The 8 genes are identified as DEGs in at least one of the four libraries (TCL1 vs TTL3; TCR2 vs TTR4; SCL5 vs SCL7; SCR6 vs STR8) by RNA-seq. In fact, some of them are genes that we are interested in. Therefore, it is not absolutely random and the “randomly” has been deleted in the manuscript. We apologize for our imprecise statement.

 

5. Line 238/239: I am not sure what you mean exactly by “exclude transcripts expressed at low levels”? Do you mean low changes in expression between the 2 conditions or do you mean low level transcripts (by low number of counts). Please clarify.

Response: We mean low changes in expression between the low-N and the control, that is Log2 (low-N treatment/control). To avoid ambiguity, we have deleted “To exclude transcripts expressed at low levels” in the manuscript.

 

6. Line 340: Autophagy might also be involved in lipid metabolism.

 Response: We also agree with you. In this study, only DEGs involved in lipid metabolism and endocytosis were detected, so only the relationship between them was discussed.

 

7. Figure 2 SCR6 vs STR8: I do not quite understand how you identified 27,000 no-DEG while at the same time you could only identify about 20,000 genes in total for STR8? Please clarify.
8. Table 3: Do you have an explanation why there are only about 20,000 genes found in STR8?

Response to comments 7 and 8: I’m very sorry, this is our clerical error. We re-checked the sequencing analysis report and it showed that the total gene number of STR8 is 30335, not 20335. Thank you very much for helping us avoiding such serious mistake.

 

9. Figure 5 and 6: The labeling is not correct for Sxx samples.

Response: “L4” has been replaced by “R4”.

10. Figure 7: You could add gene function to all genes (if known).

Response: So far, using bioinformatics methods we can only know the bioprocess these genes are probably involved in, while their specific function need further study.

 

We tried our best to improve the manuscript and made some changes in the manuscript.

We appreciate for your warm work earnestly and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestion.

 

Chunping Wang

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

in my opinion, the manuscript submitted to me for review presents very valuable research results. The use of molecular techniques in creating new and improving the existing cultivars of vegetable plants contributes to the acceleration of the breeding process. Undoubtedly, the work deserves publication in the Horticulturae Journal. However, I would suggest making a few minor adjustments:

1. The manuscript should be adapted to the requirements of the editorial office, in accordance with the applicable TEMPLATE.
2. Throughout the manuscript, replace "variety" with "cultivar".
3. I propose to shorten the title, e.g., "Comparative Transcriptome Analysis Reveals Different Low-nitrogen-responsive Genes in Pepper Cultivars".
4. Keywords should not contain words from the title. Now "pepper" and "low-nitrogen tolerance" are included in both the title and the Keywords.  But if the Authors change the title according to my remarks, instead of "pepper" you can give "Capsicum annuum L." and "low-nitrogen tolerance" can stay.
5. The Introduction is well written, but I would extend paragraphs 69-75. Please add a few more sentences regarding the usefulness of RNA-Seq for this type of research.
6. The aim of the research (L. 76-84) should be a separate paragraph.
7. L. 124-128 - place website addresses in References, and enter the reference number in the text.
8. Figure 4. descriptions are illegible.
9. Conclusion - they are written very laconically. Conclusions should be developed and completed. Write it in such a way that it can be understood without reading the entire work (e.g., there is no information here that the research concerned peppers).

After responding to my suggestions, the Editors of Horticulturae should consider publishing this manuscript.

Author Response

Dear Professor:

 We are very grateful to your comments for the manuscript. According with your advice, we amended the relevant part in manuscript. The responses to your comments are as follows.

 

1. The manuscript should be adapted to the requirements of the editorial office, in accordance with the applicable TEMPLATE.

Rresponse: Thank you very much for your kind reminding.

 

1. Throughout the manuscript, replace "variety" with "cultivar".

Rresponse: The “variety” has been replaced by “cultivar” .

 

1. I propose to shorten the title, e.g., "Comparative Transcriptome Analysis Reveals Different Low-nitrogen-responsive Genes in Pepper Cultivars".

Rresponse: According to your suggestion, it has been changed to "Comparative Transcriptome Analysis Reveals Different Low-nitrogen-responsive Genes in Pepper Cultivars".

 

1. Keywords should not contain words from the title. Now "pepper" and "low-nitrogen tolerance" are included in both the title and the Keywords.  But if the Authors change the title according to my remarks, instead of "pepper" you can give "Capsicum annuum L." and "low-nitrogen tolerance" can stay.

Rresponse: According to your suggestion, the title has been changed, and the key word “pepper” has been changed to "Capsicum annuum L.".

 

1. The Introduction is well written, but I would extend paragraphs 69-75. Please add a few more sentences regarding the usefulness of RNA-Seq for this type of research.

Rresponse: More sentences have been added to this section. “The next-generation high-throughput RNA sequencing technology (RNA-Seq) is a powerful tool for revealing genome-wide changes under biotic/abiotic stresses and can provide system level information regarding the N metabolism network. RNA-Seq analysis has been applied to the transcriptome analysis of low-N response of a single plant genotype, such as those of cucumber [25], maize [26], wheat [27], physic nut [28], and rice [29]. A large number of candidate genes involved in low-N response were detected [25,27-29]. Furthermore, the potential regulatory roles of IncRNAs in response to N stress have also been investigated [26]. However, it is difficult to reveal variations in NUE using only one genotype. Therefore, comparative transcriptome analysis of genotypes with different low N tolerances has become more recognized as a tool for understanding NUE [30-35]. A high abundance of transcripts relating to high affinity nitrate transporters (NRT2.2, NRT2.3, NRT2.5 and NRT2.6) [31] and an energy-saving assimilation pattern in N-stress tolerant Tibetan wild barley genotype have been revealed [32].”

 

1. The aim of the research (L. 76-84) should be a separate paragraph.

Response: It has been separated into a separate paragraph.

 

1. L. 124-128 - place website addresses in References, and enter the reference number in the text.

Response: These addresses has been placed in References.

 

1. Figure 4. descriptions are illegible.

Respose: Because of the large amount of information in the two groups of pictures, the handwriting should be relatively small. But this is a high-quality image. You can enlarge the whole page of Word document more than 250% to view this image.

 

1. Conclusion - they are written very laconically. Conclusions should be developed and completed. Write it in such a way that it can be understood without reading the entire work (e.g., there is no information here that the research concerned peppers).

Response: According to your suggestion, the Conclusion is more specific. “Improving NUE is urgently needed for the sustainable development of pepper production. However, present knowledge about the molecular basis governing the genetic variation of N-use efficiency (NUE) among pepper cultivars remains unclear. In this study, transcriptome of two pepper genotypes with contrary low-N tolerance (750-1, tolerant; ZCFB, sensitive) were compared using RNA-Seq and various bioinformatics methods after N starvation for 28 days. The results showed that the transcriptomic responses to low-N stress differed considerably between 750-1 and ZCFB, especially in genes that were not directly involved in N metabolism. The unique low-N-responsive genes between the two genotypes provide new insights for a comprehensive understanding of genotypic variation in NUE.”

 

We tried our best to improve the manuscript and made some changes in the manuscript.

We appreciate for your warm work earnestly and hope that the correction will meet with approval.

Once again, thank you very much for your comments and suggestion.

 

Chunping Wang

Author Response File: Author Response.docx