Metabolomic and Transcriptomic Analyses Reveal Association of Mature Fruit Pericarp Color Variation with Chlorophyll and Flavonoid Biosynthesis in Wax Gourd (Benincasa hispida)
Round 1
Reviewer 1 Report
Dear Authors,
Add more detail about both the parents their genetic background alongwith other economically important traits.
Introduction needs to be more specific. If available, add more specific references in introduction.
Author Response
Dear reviewer,
Thank you very much for your recognition of our work. Based on your suggestions, we have added the details about the lines used in this study, completed with more specific references in the introduction part and revised the language by a professor who in fluent in English in our research area. The point-by point answers are as follows:
Question 1: Add more detail about both the parents their genetic background along with other economically important traits.
Answer 1: The genetic background and other economically important traits has been added in the section “2.1 Plant Materials”. B214 was an inbred line derived from a Taiwan landrace, and was with yellow white skinned cylindrical fruit (longitudinal diametre 31 cm, diametre 12.8 cm), weighing around 2.85 kg per fruit. B227 was an inbred line derived from a cultivar ‘Sanshui Heipi Donggua’, and was with dark green skinned long cylindrical fruit (longitudinal diametre 68 cm, diametre 21.8 cm), weighing around 14 kg per fruit
Question 2: Introduction needs to be more specific. If available, add more specific references in introduction.
Answer 2: We have completed the examples of applying omics technology in underlying regulatory networks in different plant species with specific references in the introduction part.
Reviewer 2 Report
The authors have done a complete study related to fruit skin color in wax ground. In my opinion, this manuscript has high potential to publish in Agronomy journal. I have some minor comments:
- Lines 28, 29, 60: Gene name should be provided in italic format. Please apply in entire text and figures.
- Line 71: Also, scientific name have to provide in italic format. Please check the whole text.
- I could not find any supplementary data. Please add them.
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Author Response
Dear reviewer,
We quite appreciate your favorite consideration for our manuscript. We have made the revisions according to your professional suggestions and the revisions addressed are listed below:
Question 1: Lines 28, 29, 60: Gene name should be provided in italic format. Please apply in entire text and figures.
Answer 1: We have changed these gene names in italic format.
Question 2: Line 71: Also, scientific name have to provide in italic format. Please check the whole text.
Answer 2: We have checked and changed gene names in italic format in entire text and figures.
Question 3: I could not find any supplementary data. Please add them.
Answer 3: Thanks for your information. We will contact with the editor to confirm whether we have uploaded the supplementary data. If not, we will address this problem.
Reviewer 3 Report
Manuscript (agronomy-1865700) “Metabolomic and transcriptomic analyses reveal association of 2 mature fruit skin color variation with chlorophyll and flavo-3 noid biosynthesis in wax gourd (Benincasa hispida)” by Yang et al. presents an interesting study about gene expression analysis of fruit quality traits in wax gourd.
This manuscript presents a valuable study with breeding applications. However, this manuscript is very confused in some parts mainly in the description of the methodology and the experimental design. In addition, the manuscript presents important deficiencies mainly in the discussion of results. Phenotype results must be clarified. Validation of RNA-Seq through qPCR is not clear. For these reasons, this manuscript is ACCEPTABLE after a major revision.
The major points for the REVISION of the manuscript are:
Around the whole manuscript, name of genes should be in italics.
Objectives are very large. Authors must simplify the objectives not including any methodological reference in the separate paragraph. In my opinion the objectives
Plant material must be completed describing main agronomic traits of the inbred lines assayed.
Phenotyping must be clarified in the Methodology section. Data of Figure 1 must be completed with new key phenotype traits including color, sugar or acidity.
From the methodology point of view, RNA-Seq and differential expression analysis are the key methodology used. RNA-Seq characteristics must be completed, for example length of reads and nature of reads (single paired-end). In addition, biological and technical replicates must be described. Finaly the origin of the tissue analysed in both assays (qPCR and RNA-Seq) must be clarified, skin, flesh, or skin and flesh.
qRT-PCR analysis should be also clarified indicating the nature of the assayed technical and biological samples. In my opinion RNA-Samples must come from a different assay, not exactly the same that the RNA-Seq assay. This question must be clarified. In addition, the selection of the six candidate genes must be justified. If it is possible a higher number of genes to validate RNA-Seq data should increase the robustness of the experiment. A new Figure incorporating the RNA-Seq reads and qPCR results and the correlation coefficient between qPCR and RNA-Seq data must be incorporated showing all the 12 assayed genes.
Description of Results is very poor, mainly regarding phenotype analysis. The description of inbred lines is deficient.
Integration of metabolic and transcriptomic data is very poor and must be better explained in the Methodology and the Results section.
Quality of Figure 4b must be revised increasing font size.
Only two genotypes were analysed in the RNA-Seq and qPCR study, it is true? This approach is then reduced due to the limited plant material assayed. Authors must clarify this question. In addition, description of plant material must be improved with existent information about this mutation or any reference.
Discussion section is very week. Authors must clarify the novelty of the obtained results in comparison with previous transcriptome data. It is necessary to transform RNA-Seq data in biological data, this is the nature of this manuscript. However, this biological data should be discussed in term of biological information adding some biological hypothesis clarifying the development of the peach fruit shape. The election of some genes for the monitoring of this process is also very important in the Discussion section.
Conclusions are very vague. Authors must indicate the main implications of these results from an agronomical and breeding point of view.
Author Response
Dear reviewer,
Thank you very much for your efforts in reviewing our manuscript. We quite appreciate your insightful comments which for sure will help to improve the academic vigor of our manuscript. Based on your comments and requests, we have made thorough modifications on the original manuscript. Meanwhile, language of the manuscript has also been edited by a professor who is fluent in English in our research area. We hope our work could meet your standard. The detailed point by point revisions are as follows:
Question 1: Around the whole manuscript, name of genes should be in italics.
Answer 1: All the names of genes have been changed to italics in the whole manuscript.
Question 2: Objectives are very large. Authors must simplify the objectives not including any methodological reference in the separate paragraph. In my opinion the objectives
Answer 2: We have simplified the objectives of the study in the introduction part with a separate paragraph. The objective of the study is: In order to further understand the chemical components and gene regulatory network involved in the regulation of coloration in wax gourd, integrated transcriptomic and metabolomic analyses were performed using two inbred lines with distinct pericarp colors. The results would provide valuable information for understanding the complex mechanism on the coloration of wax gourd and its breeding applications.
Question 3: Plant material must be completed describing main agronomic traits of the inbred lines assayed.
Answer 3: The main agronomic economically important traits of two inbred lines have been completed as shown in the section ”2.1 Plant Materials”. B214 was an inbred line derived from a Taiwan landrace, and was with yellow white skinned cylindrical fruit (longitudinal diametre 31 cm, diametre 12.8 cm), weighing around 2.85 kg per fruit. B227 was an inbred line derived from a cultivar ‘Sanshui Heipi Donggua’, and was with dark green skinned long cylindrical fruit (longitudinal diametre 68 cm, diametre 21.8 cm), weighing around 14 kg per fruit.
Question 4：Phenotyping must be clarified in the Methodology section. Data of Figure 1 must be completed with new key phenotype traits including color, sugar or acidity.
Answer 4: In this study, we focus on the phenotype of wax gourd fruit skin color. The fruit skin color of two lines were visually distinguishable, B214 with yellow white while B227 with dark green color (Figure 1a). To determine the physiological and biochemical differences between fruit skin of these two lines, contents of chlorophyll and carotenoids were measured as described in methodology section 2.2 “Measurement of chlorophyll and carotenoid contents”. Moreover, to let the readers have a relatively clear understanding about two lines, we included other agronomically important traits including fruit shape, fruit longitudinal diametre, diametre and fruit weight. Theoretically, phenotypic traits as sugar and acidity are not necessary for the manuscript.
Question 5: From the methodology point of view, RNA-Seq and differential expression analysis are the key methodology used. RNA-Seq characteristics must be completed, for example length of reads and nature of reads (single paired-end). In addition, biological and technical replicates must be described. Finaly the origin of the tissue analysed in both assays (qPCR and RNA-Seq) must be clarified, skin, flesh, or skin and flesh.
Answer 5: Thanks for your suggestions. We have completed the RNA-Seq characteristics. For RNA-seq, the pericarp tissues of wax gourd inbred lines B214 and B227 were used as materials, each line with three biological replicates, in total 6 samples. cDNA Libraries derived from 6 samples were paired-end sequenced on the Illumina His-Seq 2500 platform following 150 bp paired-end protocol at Metware Biotechnology Co., Ltd. (Wuhan, China).
Question 6: qRT-PCR analysis should be also clarified indicating the nature of the assayed technical and biological samples. In my opinion RNA-Samples must come from a different assay, not exactly the same that the RNA-Seq assay. This question must be clarified. In addition, the selection of the six candidate genes must be justified. If it is possible a higher number of genes to validate RNA-Seq data should increase the robustness of the experiment. A new Figure incorporating the RNA-Seq reads and qPCR results and the correlation coefficient between qPCR and RNA-Seq data must be incorporated showing all the 12 assayed genes.
Answer 6: For qRT-PCR, RNA-samples were similar as that of RNA-seq but from different individuals, each inbred line with three biological replicates, each biological replicates with three technical replicates. Actually, it is 12 genes rather than 6 candidate genes were selected to validate RNA-seq data. According to your professional suggestion, we have incorporated the RNA-seq and qPCR data as well as a correlation between two data set which was illustrated in Figure 6.
Question 7: Description of Results is very poor, mainly regarding phenotype analysis. The description of inbred lines is deficient.
Answer 7: We mainly concentrate on the phenotype of wax gourd skin color and described this difference between two inbred lines by visual observation and physiological parameters, chlorophyll content and carotenoid content. The description of inbred lines has been completed in the material section.
Question 8: Integration of metabolic and transcriptomic data is very poor and must be better explained in the Methodology and the Results section.
Answer 8: The integration of metabolic and transcriptomic data has been explained in the results section 3.11 ‘Combined Analysis of Transcriptome and Metabolome’. With combined analysis, ‘Flavone and flavonol biosynthesis’ pathway is the most significantly enriched KEGG pathway, with 3 genes and 50 metabolites included in this pathway.
Question 9: Quality of Figure 4b must be revised increasing font size.
Answer 9: Thanks for your suggestion. It is true that the font size of Figure 4b is too small and we have made the change according to your suggestion.
Question 10: Only two genotypes were analysed in the RNA-Seq and qPCR study, it is true? This approach is then reduced due to the limited plant material assayed. Authors must clarify this question. In addition, description of plant material must be improved with existent information about this mutation or any reference.
Answer 10: Yes, it is true that two genotypes were analyzed in this study. The reason we choose these two inbred lines is that they were previously used for genetic mapping of pc locus controlling fruit skin color of wax gourd (Jiang et al., 2015, BMC Genomics). Actually, quite a few studies applied two genotypes for analyzing key pathways or genes regulating certain agronomically important traits in different plant species. For example, it was found that chlorophyll and related biological activity played an important role in differentiating melon stigma color by comparative transcriptome analysis using two contrasted melon lines (Lv et al., 2022, ). Zhao et al (2022) performed metabolomic and transcriptomic analysis of cucurbitacin biosynthesis in Luffa using two genotypes and provided important insights into major genes and metabolites of the cucurbitacin biosynthetic pathway, deepening the understanding of regulatory mechanisms of cucurbitacin biosynthesis in Luffa.
References:
Lv Y, Amanullah S, Liu S, et al. Comparative Transcriptome Analysis Identified Key Pathways and Genes Regulating Differentiated Stigma Color in Melon (Cucumis melo L.). Int J Mol Sci. 2022;23(12):6721.
Zhao G, Wang M, Luo C, et al. Metabolome and Transcriptome Analyses of Cucurbitacin Biosynthesis in Luffa (Luffa acutangula). Front Plant Sci. 2022;13:886870.
Question 11: Discussion section is very week. Authors must clarify the novelty of the obtained results in comparison with previous transcriptome data. It is necessary to transform RNA-Seq data in biological data, this is the nature of this manuscript. However, this biological data should be discussed in term of biological information adding some biological hypothesis clarifying the development of the peach fruit shape. The election of some genes for the monitoring of this process is also very important in the Discussion section.
Answer 11: Dear reviewer, in this manuscript, we focused on understanding the regulatory networks related to the wax gourd fruit skin color rather than peach fruit shape. We found that chlorophyll content and flavonoids are the major pigment affecting this indicated agronomic trait, and chlorophyll and flavonoids synthesis related genes varied greatly between two lines were discussed. In addition, a transcription factor in the previously mapped region was also a DEG between the two lines and this gene has a 6-bp deletion in the exon. We also discussed the probable role of this gene in regulating wax gourd fruit skin coloration. Moreover, we complemented the section with more references and discussions and hope it can reach your standard. We feel grateful for your advice.
Question 12: Conclusions are very vague. Authors must indicate the main implications of these results from an agronomical and breeding point of view.
Answer 12: Thanks for your suggestion. We have improve the conclusion.
Conclusion: In conclusion, physiological and integrated metabolic and transcriptomic analyses were performed to understand potential regulatory pathways involved in fruit color variation of wax gourd. Dark green colored inbred line B227 contained higher content of chlorophyll than yellow colored inbred line B214. 229 DAMs were identified between the two lines and flavone C-glycosides were more accumulated in B214. Key genes in chlorophyll biosynthesis pathway and flavonoid biosynthesis pathway were differentially expressed between the two lines. In addition, bHLH14 in the previously mapped pc locus controlling pericarp color of wax gourd had a higher expression in B227 than in B214. We propose that fruit coloration of wax gourd pericarp is a complex network but bHLH14 probably acts as a key factor by determining chlorophyll and flavonoid synthesis.The results provide valuable information for understanding the complex mechanism on the coloration of wax gourd and its breeding applications.
Round 2
Reviewer 3 Report
Authors have revised correctly the manuscript
