All reviews of published articles are made public. This includes manuscript files, peer review comments, author rebuttals and revised materials. Note: This was optional for articles submitted before 13 February 2023.
Peer reviewers are encouraged (but not required) to provide their names to the authors when submitting their peer review. If they agree to provide their name, then their personal profile page will reflect a public acknowledgment that they performed a review (even if the article is rejected). If the article is accepted, then reviewers who provided their name will be associated with the article itself.
Dear Authors,
I am pleased to inform you that the manuscript has improved after the last revision and can be accepted for publication.
Congratulations on accepting your manuscript, and thank you for your interest in submitting your work to PeerJ.
With Thanks
Dear Authors
The manuscript Still needs a minor revision. The authors are invited to revise the paper considering all the suggestions made by the reviewers. Please note that the requested changes are required for publication.
With Thanks
good
good
good
Review peerj-reviewing-105706-v1
The authors have addressed most of the questions quite well. The quality of the revised manuscript has been improved significantly. However, some few questions should be explained further.
For the matrix effect, one question was how to achieve matrix compounds removal for assaying accuracy. The authors’ response was relying on internal standard. That is, L-2-chlorophenylalanine was used as internal standard (0.02 mg/mL) in this study. However, for this application alone could not be sufficient to achieve the purpose. For being effective calculate the matrix effect, pure standard calibration curve and matched-matrix standard calibration curves should be performed for quantitative analysis with various spiked samples. The authors should discuss this genetic question ‘matrix compounds removal for assaying’ with reference support.
The language of the manuscript should be polished. For instance, the experiments should be described in past tense.
I commend the authors for their rigorous efforts in addressing previous feedback and significantly enhancing the quality of the manuscript titled "Integrated Transcriptome and Metabolome Analyses Reveal the Differentially Expressed Metabolites and Genes Involved in Lipid in Olive Fruits." The revised version demonstrates a substantial improvement in data presentation, analysis, and overall coherence. I am pleased to recommend the article for publication in its current form.
Experimental design is appropriate.
Results are valid.
no comment
no comment
no comment
The authors have made the changes I suggested in the last review. I recommend its publication in this journal
Dear Authors
The manuscript cannot be accepted for publication in its current form. It needs a major revision to be reconsidered for publication. The authors are invited to revise the paper considering all the suggestions made by the reviewers. Please note that the requested changes are required for publication.
With Thanks
The research significance needs further elaboration thus more readers can have their interests in reading this article.
In general it was good. However, in discussion more perspective is suggested to discuss the limitation of UPLC-MS/MS only of the results obtained.
no comment
Review peerj-105706-v0
The work contains some useful information. However, the current presentation focused too much on the very narrow topic. The research significance and general questions of this report should be discussed further.
Line 85: suggest to use ‘dietary flavonoids’ to replace ‘flavonoids’. In addition, it is suggested to describe a little bit more the health role of dietary flavonoids as the effect is achieved mainly via gut microbiota regulation. The authors can search database like Web of Science with dietary flavonoids (Title) AND health role (Title) to get reference to enhance the elaboration.
The integration of transcriptome and metabolome needs more further elaboration. For instance, one important role of integrated metabolomics is that it can reveal adaptive responses of organisms which are essential to understand the mechanism behind. The authors are suggested to search database like Web of Science with integrated metabolomics (Title) AND adaptive responses (Title) to get reference to improve the discussion.
Fruit system contains various ingredients while the UPLC-MS-MS analysis applied regular purification only. Thus, the matrix effect should be discussed. Why did the matrix compounds removal achieved successfully for obtaining satisfactory assay result? More science behind the results should be discussed. For instance, the authors can search database like Web of Science with matrix compounds removal (Title) AND assay (Title) to get reference to discuss it further.
The introduction could provide more background information on food metabolomics for quality analysis. The authors can search database like Web of Science with food metabolomics (Title) AND quality analysis (Title) to get reference to improve the introduction.
Unit: rpm is not a right unit for centrifugation. Convert it to ‘g’ in italic is needed. (Line 124, for instance).
What kind of possible approaches that can enhance the current results based on UPLC-MS-MS? Further discussion is needed as the validation was missing in this current report.
The manuscript entitled “Integrated transcriptome and metabolome analyses reveal the differentially expressed metabolites and genes involved in lipid metabolism in olive fruits” is generally well-constructed and covers important aspects of the study. However, there are some grammatical inconsistencies, awkward phrasing, and variations in verb tense that should be addressed to improve clarity and flow. Moreover, some sentences are overly complex and could be simplified or divided into shorter segments to enhance readability and overall comprehension.
In introduction section, the transition between ideas is somewhat abrupt. For instance, the text jumps from discussing olive oil consumption benefits to phenolic compounds, and then directly to increasing production methods without smooth transitions. Clearer linking sentences are required to maintain a logical flow from one topic to the next. For example, after discussing the health benefits, a sentence linking these benefits to the need for increased production would be beneficial. The manuscript also introduces various molecular mechanisms related to olive oil biosynthesis, but the introduction could more clearly highlight the specific knowledge gap the current study aims to fill. The final paragraph effectively narrows the focus to the study objectives, but this could be better aligned with the preceding information. Additionally, some claims, particularly regarding health benefits and the biosynthesis mechanisms, could be further supported by relevant references to provide a more robust literature review.
In Materials and methods section, the description of plant materials and transcriptome analysis is somewhat vague. Although the study references previous work for plant growth conditions and nanopore sequencing, the details should be briefly summarized here for completeness and to ensure this manuscript can stand alone without requiring the reader to consult other papers.
There is also an abrupt shift between transcriptomic data acquisition and the analysis methods (lines 114-116), and this could be smoothed out by adding a linking sentence explaining how the data fits into the current study. In the Detection of Untargeted Metabolites section, the protocol is generally clear, but some steps lack specificity. For instance, the exact temperature or conditions for the sonication process are missing, which could introduce variability in replication attempts. Additionally, specify the type of grinder and add temperature used during centrifugation. The Data Processing section is mostly sound, but the sentence structure is awkward in places.
Additionally, while the mention of software and databases used for analysis is helpful, a brief description of how the correlation network between differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) was constructed would provide further clarity to the experimental approach. It is also important to clearly explain why the chosen thresholds for significance were appropriate for this study.
The results presented are comprehensive and relevant, particularly given the detailed metabolomic profiling of JZ and KLD fruits using UPLC-MS/MS. The detection of 725 metabolites across 47 classes adds depth to the study. However, the claim of increased acetyl-CoA and decanoic acid content should be backed up by statistical analysis to solidify conclusions regarding metabolic differences. The comparison between JZ and KLD fruit metabolites is valid, but more specific statistical evidence is necessary to strengthen the argument for significant metabolite differences, especially given variability among replicates of KLD noted in the heatmaps (Figure 2D). Additionally, the use of PCA and OPLS-DA for multivariate analysis provides a robust framework for data interpretation. Yet, further statistical support, like ANOVA or pairwise comparison tests, would bolster the findings and their significance.The manuscript could benefit from more focused discussions on the biological significance of these pathways (Fig. 3), especially those with implications in lipid metabolism and the biosynthesis of specific lipid compounds. There is an opportunity to better integrate the results of KEGG enrichment with broader metabolic shifts observed in the fruits. Additionally, statistical methods applied in the OPLS-DA model are reported with values of R2X = 0.575, R2Y = 0.993, and Q2 = 0.965, indicating a well-fitting model. These results suggest strong predictive power and low overfitting, enhancing the reliability of the dataset. However, cross-validation results or permutation tests could be included to further substantiate these claims.
The discussion focuses on describing the metabolic pathways and gene expression differences, but it lacks a deeper exploration of the biological significance of these findings. The manuscript would benefit from a discussion on the potential agronomic or commercial implications of these variations, particularly in the context of olive oil production and quality. The differences between JZ and KLD are highlighted, but there is limited comparison to other olive varieties or to general trends in olive lipid metabolism. Integrating findings from other studies (beyond the cited references) could strengthen the generalizability of the findings.
Integrate the biological relevance of the observed metabolite shifts. For example, highlight the significance of increased sphingolipid content in KLD fruits or delve into the implications of altered fatty acid metabolism in olive fruit development and maturation.
The discussion around specific metabolic pathways (e.g., TCA cycle, sphingolipid metabolism, and arachidonic acid metabolism) could be more detailed. While the main findings are mentioned, there is room for more discussion on how these pathways might interact and contribute to lipid regulation in a broader sense.
The PCA and OPLS-DA results are mentioned as being reliable, but the interpretation of the statistical model could be explained more clearly. For example, explaining why the high R2Y value indicates model reliability and discussing any potential limitations of these statistical approaches in metabolomics would enhance the manuscript’s depth.
The conclusion lacks a statement on the next steps for research. Suggesting further investigations, such as functional validation of the identified DEGs or exploration of these pathways in other olive varieties, would add a forward-looking perspective. The practical significance of these findings for olive breeding, oil production, or genetic engineering is not addressed. Discussing how these results could be applied to improve lipid content or composition in olives would make the conclusion more impactful.
Specific comment:
(Line 42) "Olive oil is one the most important" where "one of the most important" should be used.
The statement (Lines 151-152) "a total of 79 lipid compounds were detected in olive fruits, counting 10.9% of 725 metabolites" can be rephrased to emphasize the significance of lipid diversity, e.g., "Among the 725 detected metabolites, 79 (10.9%) were lipid compounds, showcasing a diverse lipidome that encompasses fatty acyls, prenol lipids, and sphingolipids."
In sentences describing correlation analysis (Lines 155-157), specificity regarding statistical methods used (e.g., Spearman/Pearson correlations) would improve clarity. Also, the discussion around volcano plots and KEGG enrichment (Lines 160-171) could be more concise, emphasizing the biological and metabolic relevance of the upregulated and downregulated metabolites.
The phrase in Lines 137-138 "significant difference was determined by thresholds of FC > 1, P < 0.05, and VIP > 1" could be rephrased to “A significant difference between metabolites was considered when the fold change (FC) exceeded 1, P-value was less than 0.05, and the variable importance in projection (VIP) score was greater than 1.” This revision improves readability and technical clarity.
(Lines 200-202) “In our study, the contents of acetyl-CoA and decanoic acid were increased in KLD fruits compared to JZ fruits (Figure 2D).” Upon visual inspection of the heatmap in Figure 2D, it becomes evident that one of the replicates for KLD exhibits a lower acetyl-CoA content. This variation highlights the need for a thorough statistical analysis to assess the significance of these differences. Without such an analysis, it is difficult to draw robust conclusions. I strongly recommend the application of appropriate statistical tests to quantify the variation and validate the observed trends, ensuring that the results can be confidently presented to the scientific community.
"Acetyl-CoA participates in several metabolic pathways, such as TCA cycle, fatty acid biosynthesis, fatty acid elongation, fatty acid degradation, and fatty acid metabolism." (Lines 273-275), this could be revised to: "Acetyl-CoA is involved in several metabolic pathways, including the TCA cycle, fatty acid biosynthesis, elongation, degradation, and overall fatty acid metabolism."
Certain phrases lack clarity, such as (Lines 256-257), "greater PC1 value indicated the higher degree of genetic variation." This should be more specific to whether this refers to genetic or metabolic differences.
Lines 316-323 “Our results indicate that arachidonic acid metabolism and limonene degradation directly affect the quality and quantity of lipid metabolites in JZ and KLD…….” However, the manuscript would benefit from a more in-depth analysis of these findings, especially regarding the biological and metabolic implications of these DEG expression patterns. Please expand on how the upregulation of decanoic acid-related DEGs and the downregulation of those involved in leukotriene D4 and perillyl alcohol degradation may influence lipid metabolism in KLD, and discuss these trends in the context of current literature on lipid metabolic pathways. A deeper exploration of the mechanistic link between these gene expression changes and lipid metabolite variations would greatly enhance the clarity and impact of the results.
Provide complete data that was obtained during research work and used in the correlation, orthogonal
136 partial least square-discriminant analysis, expression pattern analysis in supplemental data files.
While the experimental design is solid, but providing additional statistical validation, and offering deeper insights into the biological significance of the findings would significantly enhance the manuscript.
The study presents valuable findings but would benefit from more thorough analyses and clearer communication of the broader implications. Moreover, raw data is needed to judge the validity of findings in supplemental files.
No comment
No comment
No comment
The manuscript presents a valuable contribution to the understanding of lipid metabolism in olive fruits. By employing untargeted metabolomics and RNA-Seq analyses, the authors have successfully identified differentially accumulated metabolites and expressed genes associated with lipid synthesis and metabolism in two olive cultivars with contrasting oil content.
-Comments and Suggestions for Authors
- Abstract
-While the abstract mentions the identification of 38 lipid compounds, it would be beneficial to provide some quantitative information about their relative abundance or differences between the two cultivars.
- Introduction
- While the introduction is generally well-written, some sections could be clarified to improve readability and understanding. For example, the discussion of the TCA cycle and its role in fatty acid biosynthesis could be further elaborated.
-The introduction might benefit from a more specific focus on the unique aspects of lipid metabolism in olive fruits, rather than providing a general overview of fatty acid metabolism in plants.
-The introduction could more explicitly highlight the research gap that this study aims to address, emphasizing the lack of clarity regarding the molecular mechanisms underlying lipid biosynthesis and metabolism in olive cultivars.
- Materials and Methods
- This section provides a clear and concise overview of the plant materials, stage selection, and data source for the transcriptome analysis.
- The authors mentioned the stage (MI-3), it would be beneficial to clarify if there were any specific selection criteria for the 108 fruits used (e.g., size, maturity within the stage).
Results
-Overall, this results section provides a solid foundation for understanding the metabolic differences between JZ and KLD olive fruits.
Discussion
- In discussion section, the authors mention DEGs associated with lipid metabolites, it would be beneficial to provide more specific information about the genes involved and their potential functions.
- The discussion could be strengthened by further elaborating on the biological significance of the identified DEGs and their potential roles in regulating lipid metabolism.
- The discussion could benefit from a more in-depth comparison with previous studies on olive fruit metabolomics, especially those focusing on lipid metabolism.
All text and materials provided via this peer-review history page are made available under a Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.