The Combined Analysis of Transcriptome and Metabolome Provides Insights into Purple Leaves in Eruca vesicaria subsp. sativa
, Xiaofeng Li 1,†, Changwei Zhang 2, Lu Gao 1, Yuying Zhu 1, Shiwei Wei 3, Ying Li 2, Mingmin Jiang 4, Hongfang Zhu 1,* and Zhaohui Zhang 1,5,*
Citlali Fonseca-GarcÃa
Round 1
Reviewer 1 Report
The research (The combined analysis of transcriptome and metabolome provides insights into purple leaves in Eruca vesicaria subsp. sativa) studied mechanisms, transcriptomic and metabolomic properties of purple leaf coloration of Eruca vesicaria subsp. Sativa is an essential oil crop of cruciferous species. The manuscript idea was interesting, however, the manuscript methods are missing many details also the manuscript needs English and grammar revision.
Major revision:
1. The author needs to mention the seed sources.
2. In the greenhouse experiment, the authors must include the growth conditions, temperature, humidity, type of soil, and chemical and physical soil properties.
3. Also the irrigation rate and the field capacity need to include.
4. The authors mentioned in the introduction that (anthocyanins, phenolic compounds, a group of flavonoids, and responsible for coloring pigments in leaves, flowers, and fruits). Why didn’t they measure it in leaves?
5. Purple leaves pigments need to analysis and included in the manuscript.
6. Discussion part is poor and needs to update with informations.
7. The manuscript needs English and grammar revision
Author Response
Point 1:The author needs to mention the seed sources.
Response: Arugula seeds were bought from Nantong Shengxiang Agricultural Technology Co., Ltd, which has been added in Part 2.1.
Point 2: In the greenhouse experiment, the authors must include the growth conditions, temperature, humidity, type of soil, and chemical and physical soil properties.
Response: Thanks. We have added growth conditions in part 2.1.
Point 3: Also the irrigation rate and the field capacity need to include.
Response: We have added relevant information in part 2.1.
Point 4: The authors mentioned in the introduction that (anthocyanins, phenolic compounds, a group of flavonoids, and responsible for coloring pigments in leaves, flowers, and fruits). Why didn’t they measure it in leaves?
Response: We totally detected 747 metabolites, including phenolic acids, flavonoid, flavonols and other phenolic compounds (Figure 1b). We obtained 84 differentially expressed metabolites including 5 anthocyanins and other flavonoids. The top4 with largest increase in content were anthocyanins. And considering anthocyanins are the main pigments responsible for purple color, we mainly focused on the difference of anthocyanin contents between the purple leaves and green leaves and analyzed the DEGs regulating anthocyanin biosynthesis.
Point 5: Purple leaves pigments need to analysis and included in the manuscript.
Response: Thanks a lot. We have added discussion in Part4.
Point 6: Discussion part is poor and needs to update with informations.
Response: We have revised the discussion part. Thanks for your advice.
Point 7: The manuscript needs English and grammar revision.
Response: Thank you very much. We carefully revised English and grammer and hope it is acceptable.
Reviewer 2 Report
The following is a review of the manuscript from Xi et al. “The combined analysis of transcriptome and metabolome provides insights into purple leaves in Eruca vesicaria subsp. sativa” for Agronomy.
In this manuscript, the mechanisms to form the two leaf colors of a purple arugula cultivar and a green arugula with purple and green leaves, respectively were analyzed using metabolomics and transcriptomics analysis. It is the opinion of this reviewer that there are not sufficient novel data or discussion to publish these results in their current form. The results do not have a deep description of the data, their metabolomics and transcriptomics analysis provide a lot of information that could be described with more detail. The authors do not provide any type of validation of their in silico analysis such as RT-qPCR. In addition, the materials and methods were not described properly, there is not enough information to replicate the experiments and data analysis. In conclusion, the current manuscript lacks a good description and discussion of the metabolomics and transcriptomics data presented here.
Specific comments I have:
Abstract. Highlight the importance of the study in this section, why is important to study the color of the leaves?
L24. Change to depleted and enriched metabolites.
Material and methods. There is not enough information to replicate the experiments.
Sections 2.3 and 2.4. Conditions, number of replicates, metabolite extraction conditions, chromatography + mass spectroscopy? sequencing platform, software, codes, etc. to replicate the metabolites detection, transcriptomic sequencing, and data analysis.
Results. There is not a deep description of the data. What is CK and PA? Define.
L137. Change metabolomic sequencing by metabolomics analysis.
L142. Change sequencing by detection.
Figure 1 legend: (b) duplicated. Indicate meaning of CK and PA.
L189. scientific names in italics
Sections 3.3 and 3.4. same title!
Figure 5. Describe more the a) Nine quadrants flat.
Discussion. The discussion could be extended. For example, discuss the data related to enriched metabolic pathways and GO terms. You can discuss more about the seven structural genes and other data found in the combined metabolomic and transcriptomic analyses, etc.
L280. References of the previous studies.
Author Response
Point 1: Abstract. Highlight the importance of the study in this section, why is important to study the color of the leaves?
Response: Thanks for your comment. We have revised the importance of the study in abstract.
Point 2: L24. Change to depleted and enriched metabolites.
Response: Thanks for your recommendation. We have made revisions.
Point 3: Material and methods. There is not enough information to replicate the experiments.
Sections 2.3 and 2.4. Conditions, number of replicates, metabolite extraction conditions, chromatography + mass spectroscopy? Sequencing platform, software, codes, etc. to replicate the metabolites detection, transcriptomic sequencing, and data analysis.
Response: Thanks for your kind suggestion. We have added information in method part
Point 4: Results. There is not a deep description of the data. What is CK and PA? Define.
Response: CK represents green arugula and PA represents purple arugula. To reduce misunderstanding, CK was changed to GA in manuscript.
Point 5: L137. Change metabolomic sequencing by metabolomics analysis.
L142. Change sequencing by detection.
Figure 1 legend: (b) duplicated. Indicate meaning of CK and PA.
L189. scientific names in italics
Sections 3.3 and 3.4. same title!
Response: Thank you for pointing out these mistakes. We have made corresponding revisions.
Point 6: Figure 5. Describe more the a) Nine quadrants flat.
Response: Thanks. We added corresponding description in Figure 5 legend.
Point 7: Discussion. The discussion could be extended. For example, discuss the data related to enriched metabolic pathways and GO terms. You can discuss more about the seven structural genes and other data found in the combined metabolomic and transcriptomic analyses, etc.
Response: Thanks for your constructive suggestions. Relevant discussion have been added.
Point 8: L280. References of the previous studies.
Response: Related references have been added. Thanks a lot.
Reviewer 3 Report
The manuscript entitled "The combined analysis of transcriptome and metabolome provides insights into purple leaves in Eruca vesicaria subsp.sativa" is generally interesting and suitbale for the journal. The aim of the work is clear and the methodologies together with the results and discussion part are clearly reported. The results are well presented and supported by a sufficient number of references.
The overall manuscript can be considered suitbale for the publication, however I suggest
1) to add a list of abbreviations
2) In the figure 4a,b check size and font
Author Response
Point 1: Add a list of abbreviations
Response: Thank you very much. We have added the abbreviation list following Part 5.
Point 2： In the figure 4a, b check size and font
Response: Thanks for your reminding. The size and font of figure 4 have been revised.
Round 2
Reviewer 1 Report
The authors gave sufficient answers and corrected the manuscript, so I recommend accepting it.
Author Response
Point 1：The authors gave sufficient answers and corrected the manuscript, so I recommend accepting it
Thanks very much for your kind word. Your comments and suggestions are valuable and improve our manuscript quality, thank you once again.
Reviewer 2 Report
The authors addressed the suggestions and comments made by this reviewer. However, from my perspective, the discussion section could be more extended. I recommend to correlated more the GO and KEGG data. For example, the GO term phenylalanine aminotransferase with 20 genes was enriched, a key enzyme of an alternative phenylalanine (Phe) synthesis pathway from phenyl pyruvate to Phe (Yoo, et al., 2013), the key precursor of anthocyanins as well. That founding was supported by a KEGG enrichment of Phe biosynthesis, Phe metabolism, biosynthesis of amino acids, and Anthocyanin biosynthesis (all of them with an orange q-value)…..
Yoo, H., Widhalm, J., Qian, Y. et al. An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase. Nat Commun 4, 2833 (2013). https://doi.org/10.1038/ncomms3833
In addition, the authors did not consider my comment related to provide a type of validation of their in silico analysis such as RT-qPCR. Why do they consider that it is not necessary to validate?
Other specific comments:
Figure 4. q-value not p-value. The analysis has p-adjusted values.
L275. Phenylalanine ammonia lyase, add space.
L307. Leaf formation? modify.
Author Response
Point 1: The authors addressed the suggestions and comments made by this reviewer. However, from my perspective, the discussion section could be more extended. I recommend to correlated more the GO and KEGG data. For example, the GO term phenylalanine aminotransferase with 20 genes was enriched, a key enzyme of an alternative phenylalanine (Phe) synthesis pathway from phenyl pyruvate to Phe (Yoo, et al., 2013), the key precursor of anthocyanins as well. That founding was supported by a KEGG enrichment of Phe biosynthesis, Phe metabolism, biosynthesis of amino acids, and Anthocyanin biosynthesis (all of them with an orange q-value)…..
Yoo, H., Widhalm, J., Qian, Y. et al. An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase. Nat Commun 4, 2833 (2013). https://doi.org/10.1038/ncomms3833
Response：Thanks for your kind advice. We have added related discussions which we hope is acceptable.
Point 2: In addition, the authors did not consider my comment related to provide a type of validation of their in silico analysis such as RT-qPCR. Why do they consider that it is not necessary to validate?
Response：We are very sorry about this. We would like to provide qRT-PCR results. However, the culture time of arugula seedlings were less than 20 days at present and only 7 days were allowed for the revision. In our manuscript, 20-day seedlings were used for transcripomics and metabolomics. Although without qT-PCR test, we considered that the transcriptome data was credible as transcriptome sequencing has been widely used in the study of various species with the development of sequencing technology.
Point 3: Other specific comments:
Figure 4. q-value not p-value. The analysis has p-adjusted values.
L275. Phenylalanine ammonia lyase, add space.
L307. Leaf formation? modify.
Response：Thanks for pointing out these mistakes. We have made corrections according to your comments.
