Integrating Transcriptomics and Metabolomics Analyses to Reveal the Potential Molecular Mechanism of Citrus junos Aroma Enhancement by Protected Cultivation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study investigates protective cultivation practices, such as plastic greenhouses, to enhance the aroma of yuzu (Citrus junos cv. ‘Kitou’). It compares field-grown (CJKTF) and greenhouse-grown (CJKTP) plants, using sensory and molecular analyses to evaluate differences in aroma and volatile organic compounds (VOCs). Integrating metabolomic (GC-MS) profiling and transcriptomic analysis, the research provides insights into the biochemical and genetic factors influencing aroma variation. The identification of specific VOCs and terpene synthase genes marks a valuable contribution to aroma-related crop quality improvement. The manuscript has been critically reviewed, and suggestions for improvement in several sections are outlined in detail below.

 

Line no. 35: The scientific name Citrus reticulata should be fully written the first time it appears in the text and can be abbreviated as C. maxima for subsequent mentions.

 

Line no. 103: The Materials and Methods section should include information about the sensors and their specific substances to help readers understand the origin and relevance of the data. Additionally, the 'Electronic Nose Analysis' subsection should be expanded to provide detailed information about each sensor and its performance specifications.

 

Figure 2: Please clarify what "CSCXF" refers to in the figure legend. Do you mean to write "CJKTF" instead? If CSCXF is indeed different from CJKTF, please explain what it represents in the legend to avoid confusion.

 

Table 1: Terpenoids, especially monoterpenes and sesquiterpenes, are vital for the characteristic aroma of citrus fruits. Notably, most terpenoids are downregulated in CJKTP compared to CJKTF, as shown in the table. In the Results or Discussion section, please explain how this reduction may affect the aroma profile of CJKTP. If there are terpenoids highly expressed in CJKTP, discussing their roles in enhancing yuzu's aroma would strengthen your paper. Please support this with relevant literature, particularly for significantly enriched terpenoids.

Additionally, limonene is a key contributor to citrus aroma. Did you observe limonene or its derivatives in your data? Highlighting their presence and relevance to the aroma profile of CJKTP would be valuable.

Line no. 161: As mentioned, rOAV is crucial for identifying key flavor compounds and helps to clarify the contribution of each aroma component. Figure 3 illustrates the log rOAV values in CJKTP and CJKTF. However, if the conclusion is that the aroma is improved in CJKTP compared to CJKTF, it raises a question about why many VOCs have higher log rOAV values in CJKTF. Could you please clarify this point? If the log rOAV is indeed higher in CJKTF, does that indicate a stronger aroma in CJKTF? This appears to contradict the overall conclusion and would benefit from further explanation.

Line no. 173: Please specify the test and control samples used for the transcriptomic and metabolomic analyses. This information should be clearly detailed in the manuscript to ensure clarity.

Line no. 244:  Kamiloglu (2011) is not cited properly in the manuscript.

Line no. 346: The manuscript discusses the biosynthesis pathways of terpenes and diterpenes; however, it does not include a list of differentially expressed genes (DEGs) related to these pathways. Including a supplementary table that lists these DEGs along with their log fold changes would be beneficial. This addition would support your findings and provide valuable reference data for future studies.

Lines 348–374: For this section as well, including a table that lists the DEGs, along with their gene descriptions and log fold changes, would help readers understand which genes are significantly expressed and how they contribute to the changes in the discussed pathways.

Lines 403–405: There is an inconsistency in how time is abbreviated. In a previous section, "minutes" is written in full, while in this section, it is abbreviated as "min." Please maintain consistency throughout the manuscript regarding abbreviations.

Line no. 422: Write the scientific name Citrus sinensis in italics.

Line no. 429: Please correct the abbreviation to differentially expressed genes (DEGs).

 

 

Author Response

1. Line no. 35:The scientific name Citrus reticulatashould be fully written the first time it appears in the text and can be abbreviated as C. maxima for subsequent mentions.

Response 1: Thanks for the suggestion. We have made the revision. Please see lines 35-36.

2. Line no. 103: The Materials and Methods section should include information about the sensors and their specific substances to help readers understand the origin and relevance of the data. Additionally, the 'Electronic Nose Analysis' subsection should be expanded to provide detailed information about each sensor and its performance specifications.

Response 2: Thank you for your valuable suggestion. We have added the information related to e-nose sensors and their specific substances in the Materials and Methods section. In addition, we have also added information about the details related to electronic nose analysis. E-nose system equipped with 10 metal oxide semiconductor sensors: W1C, primarily sensitive to aromatic components and benzene compounds; W5S, primarily sensitive to nitrous oxides compounds; W3C, primarily sensitive to ammonia and aromatic components; W6S, primarily sensitive to hydrocarbons; W5C, primarily sensitive to alkanes, aromatics, and small polar compounds; W1S, primarily sensitive to a broad range of methane compounds; W1W, primarily sensitive to inorganic sulfides and many terpenes; W2S, primarily sensitive to most alcohols, aldehydes, and ketones; W2W, primarily sensitive to aromatics and organic sulfides; W3S, primarily sensitive to long-chain alkanes (see lines102-109). The response value for each sensor is G/G0, where G and G0 are the resistance of the sensor when exposed to sample volatiles and clean air, respectively. Data were collected from 57 to 60 sec steady-state response values for each test and analyzed using WinMuster (version 1.6.2) software (see lines 111-115). These differentially accumulated VOCs comprised 12 terpenoids, 9 esters, 7 ketones, 5 heterocyclic compounds, 3 organic acids, and one each of alcohol, sulfur compound, ether, aldehyde, and hydrocarbon, of which 16 of these belong to the aromatic compounds. (Figure 2D; Table 1). There were more aromatic and terpenes compounds differentiators between CJKTP and CJKTF, and consistent with the results of the electronic nose analyses, the W2W and W1W sensors, which are primarily sensitive to aromatic and terpenes compounds, were the main contributors to the organoleptic differences in the aromas of CJKTP and CJKTF (Figure 1D) (lines223-230).

3. Figure 2: Please clarify what "CSCXF" refers to in the figure legend. Do you mean to write "CJKTF" instead? If CSCXF is indeed different from CJKTF, please explain what it represents in the legend to avoid confusion.

Response 3: Thank you very much for your careful review. This is an error. We have changed CSCXF to CJKTF.

4. Table 1: Terpenoids, especially monoterpenes and sesquiterpenes, are vital for the characteristic aroma of citrus fruits. Notably, most terpenoids are downregulated in CJKTP compared to CJKTF, as shown in the table. In the Results or Discussion section, please explain how this reduction may affect the aroma profile of CJKTP. If there are terpenoids highly expressed in CJKTP, discussing their roles in enhancing yuzu's aroma would strengthen your paper. Please support this with relevant literature, particularly for significantly enriched terpenoids.

Additionally, limonene is a key contributor to citrus aroma. Did you observe limonene or its derivatives in your data? Highlighting their presence and relevance to the aroma profile of CJKTP would be valuable.

Response 4: Thank you very much for your insightful suggestions. We provide relevant explanations and clarifications in the discussion section. Please see lines 384-394. Volatile terpenes not only give off an aroma but many also have the ability to repel or even kill pests. Consistent with the above hypothesis, all terpenes with reduced levels in CJKTP have biopesticide properties (Table 1). This results in more terpenes with higher levels in CJKTF, which seems to contradict the stronger aroma of CJKTP. However, the formation of odor is a complex system and is the result of a combination of VOC types and contents, and the odor components may interact with each other. There are four modes of interaction in the odor complex: integration, synergism, antagonism, and independence; this suggests that the odor outcome cannot be determined by the accumulation of VOC content alone [51]. The effect of changes in certain substances on the overall odor is not clear from this study. What is certain is that the overall odor quality of CJKTP is better than that of CJKTF.

Limonene is indeed a major contributor to citrus aroma, and it is higher in CJKTP compared to CJKTF, but not significantly (see Table S1). Therefore, it was not given more attention in this study.

5. Line no. 161: As mentioned, rOAV is crucial for identifying key flavor compounds and helps to clarify the contribution of each aroma component. Figure 3 illustrates the log rOAV values in CJKTP and CJKTF. However, if the conclusion is that the aroma is improved in CJKTP compared to CJKTF, it raises a question about why many VOCs have higher log rOAV values in CJKTF. Could you please clarify this point? If the log rOAV is indeed higher in CJKTF, does that indicate a stronger aroma in CJKTF? This appears to contradict the overall conclusion and would benefit from further explanation.

Response 5: Thank you for raising this important point. In this study, we calculated the corresponding rOAV based on the available sensory thresholds for some compounds. rOAVs for some substances are currently unavailable. Judging overall odor strength based on the rOAV is incomplete. Even so, we found that the total rOAV of CJKTP (5.1×10⁷) is higher than that of CJKTF (4.8×10⁷) (see lines 240-242).

6. Line no. 173: Please specify the test and control samples used for the transcriptomic and metabolomic analyses. This information should be clearly detailed in the manuscript to ensure clarity. Line no. 244:  Kamiloglu (2011) is not cited properly in the manuscript.

Response 6: Thank you for your detailed comments. we have described the test and control samples for the transcriptome and metabolome (see lines 216-217, 261). The Kamiloglu (2011) literature has been correctly cited (see lines 324).

7. Line no. 346: The manuscript discusses the biosynthesis pathways of terpenes and diterpenes; however, it does not include a list of differentially expressed genes (DEGs) related to these pathways. Including a supplementary table that lists these DEGs along with their log fold changes would be beneficial. This addition would support your findings and provide valuable reference data for future studies. Lines 348–374: For this section as well, including a table that lists the DEGs, along with their gene descriptions and log fold changes, would help readers understand which genes are significantly expressed and how they contribute to the changes in the discussed pathways.

Response 7: Thank you very much for your professional opinion. We have added information about DEGs in terpenoid and flavonoid synthesis pathways, including log fold changes and gene descriptions in Table S2.

8. Lines 403–405: There is an inconsistency in how time is abbreviated. In a previous section, "minutes" is written in full, while in this section, it is abbreviated as "min." Please maintain consistency throughout the manuscript regarding abbreviations. Line no. 422: Write the scientific name Citrus sinensisin italics. Line no. 429: Please correct the abbreviation to differentially expressed genes (DEGs).

Response 8: Thank you very much for your careful review. We have made the corresponding revisions (see lines 120-121, 125, 148, 154).

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for providing me with the opportunity to review this article titled as Integrating transcriptomics and metabolomics analyses to reveal the potential molecular mechanism of citrus junos aroma enhancement by protected cultivation.

In this study authors described that qroma is an important flavour that assesses the quality of yuzu. In this study, C. junos 14 cv. ‘Kitou’ grown in open field (CJKTF) and plastic greenhouses (CJKTP), respectively, were se- 15 lected as the study material. Significant differences in aroma performance between CJKTF and 16 CJKTP were found by the olfactory senses of the members of this research group and the electronic 17 nose, with CJKTP having a stronger aroma. Regarding VOCs, GC-MS analyses revealed 13 VOCs 18 up-regulated and 28 VOCs down-regulated in CJKTP compared to CJKTF. Transcriptome analysis 19 revealed that 515 genes were up-regulated and 720 genes were down-regulated in CJKTP compared 20 to CJKTF. The differential VOCs nerolidol and γ-cadinene, and the differential genes nerolidol syn- 21 thase 1 (NES1), nerolidol synthase 1-like (NES1-like), and cadinene synthase (DCS) were in the sesquiter- 22 pene synthesis pathway and showed significant correlation. NES1, NES1-like, and DCS encode ter- 23 pene synthases, which may be involved in the biosynthetic pathway of nerolidol and γ-cadinene. In 24 conclusion, the use of plastic greenhouses for cultivation may improve the quality and aroma inten- 25 sity of yuzu, as well as alter the expression of related genes, compared to field cultivation. These 26 results suggest that protected cultivation is a suitable cultivation practice to enhance the aroma of 27 yuzu.

My observations: This study is important and informative. I recommend minor revisions.

1. Authors should add more details about yuzu in introduction section special emphasize on its nutritional components.
2. Please submit genomics and proteomics data in suitable repository and add its link into the revised version (important)
3. If possible, please add more details about the gene interaction to elucidate molecular mechanisms in depth.
4. Add company model and number of all the instruments.
5. Elaborate discussion and conclusion section.
6. Refence and typographical errors should be double checked.

Author Response

1. Authors should add more details about yuzu in introduction section special emphasize on its nutritional components.

Response 1: Thank you very much for your expert advice. We have added details about the nutritional aspects of yuzu in the introductory section. Please see line 42-46.

2. Please submit genomics and proteomics data in suitable repository and add its link into the revised version (important)

Response 2: Thank you very much for your professional opinion. We have deposited the omics data on the NCBI website. Please see lines 499-501.

3. If possible, please add more details about the gene interaction to elucidate molecular mechanisms in depth.

Thank you very much for your valuable suggestion. This study primarily focuses on the effects of protected cultivation on improving the aroma quality of yuzu and explores the preliminary molecular regulatory mechanisms involved. We fully agree that a more in-depth investigation into the gene interactions and regulatory networks would provide a deeper understanding of the molecular basis underlying these effects. As such, we acknowledge this as an important direction for future research and have accordingly added a statement in the revised manuscript (see lines 423–424) to emphasize the need for further studies on detailed molecular interactions.

4. Add company model and number of all the instruments.

Response 4: Thank you very much for your suggestion. We have added the company model numbers and numbers of all instruments in the Material Methods section.

5. Elaborate discussion and conclusion section.

Response 5: Thank you very much for your suggestion. We have revised the discussion and conclusion sections.

6. Refence and typographical errors should be double checked.

Response 6: Thank you for your attention to detail. We checked for references and typographical errors.

Reviewer 3 Report

Comments and Suggestions for Authors

The article addresses an important topic at the intersection of biotechnology and horticulture. The results are potentially valuable for the practice of growing citrus junos. The authors combined transcriptomics and metabolomics, which allowed them to comprehensively investigate the effects of protected cultivation on the aroma quality of the fruits studied.  The paper identified 904 volatile compounds, 41 of which were shown to be differential for growing conditions. Analyses and correlations were performed and presented in a professional manner. The research can be applied to orchard practice - the effect of growing conditions on fruit quality. However, the article needs several corrections:

1. There is a lack of a clearly stated research hypothesis in the final part of the introduction.
2. There is a lack of a clearly formulated research question: the article indicates the purpose of the study, but does not define a specific scientific problem.
3. The research methodology chapter is missing.
4. E-nose analysis was performed on a panel of 9 people, but no statistical analysis of sensory data is given. State exactly how long the analysis took, incubation conditions, standardization.
5. The validation of gene expression results is missing
6. Figures and figures are not very clear. Add larger axis descriptions and legends, especially in fig. 1C-1E. and fig. 2A-2F.
7. In Table 2, for better clarity, sort the data by log2FC or VIP
8. Conclusions are too vague. They should indicate specific practical and research implications and make a suggestion for future research.
9. Little critical attention to the limitations of the study. For example: the authors did not mention whether the change in the VOC profile could have been a result of fruit maturity or microclimatic differences other than just crop structure.

Author Response

1. There is a lack of a clearly stated research hypothesis in the final part of the introduction.

Response 1: Thank you for your insightful suggestion. We have added the research hypothesis in the introduction section. Compared with conventional open-field cultivation, protected cultivation offers improved sustainability by mitigating climatic risks and promoting more efficient utilization of resources such as fertilizers, pesticides, and water. It also facilitates enhancements in crop yield and quality. This approach has been successfully applied in various crops to enhance desirable traits; for example, supplemental lighting under protected conditions has been shown to improve the aroma and quality of grape berries. Therefore, the use of protected cultivation may be promising for improving the quality of yuzu aroma. Please see lines 75-76.

2. There is a lack of a clearly formulated research question: the article indicates the purpose of the study, but does not define a specific scientific problem.

Response 2: Thank you for your valuable comment. We redefined the specific scientific question in the introductory section. That is, how protected cultivation affects citrus aroma and its underlying molecular mechanisms (see lines 77-78).

3. The research methodology chapter is missing.

Response3: Thank you for your suggestion. Following the formatting requirements of the journal, we have moved Material Methods to the correct location (see lines 89-154).

4. E-nose analysis was performed on a panel of 9 people, but no statistical analysis of sensory data is given. State exactly how long the analysis took, incubation conditions, standardization.

Response 4: Thank you for pointing this out. Regarding the methodology of the electronic nose analysis, we have updated it in the Materials and Methods section (see lines 100-115). The sensory analysis was conducted for a group of 9 people, who were non-professional odor evaluators. We aimed to simply and rudely evaluate the yuzu aroma from the consumer's point of view. Therefore, there are no strict statistics and criteria for this part of the results.

5. The validation of gene expression results is missing

Response 5: Thank you very much for your professional advice. It is important to verify the gene expression by qPCR in this study. However, limited by the lack of samples and the long growth cycle of fruit trees, it is difficult to supplement the relevant experiments in a short time. Next-generation transcriptome sequencing has developed to be a very sophisticated technology and has been very accurate in gene quantification. In this study, the RNA-seq analysis was able to show the gene expression with high accuracy. In addition, we will continue to investigate the related genes in this study in the future.

6. Figures and figures are not very clear. Add larger axis descriptions and legends, especially in fig. 1C-1E. and fig. 2A-2F.

Response 6: Thank you for your careful review. We have revised Figure 1 as well as the legend for Figures 1, 2.

7. In Table 2, for better clarity, sort the data by log2FC or VIP

Response 7: Based on your suggestion, we re-sorted the data in Table 1

8. Conclusions are too vague. They should indicate specific practical and research implications and make a suggestion for future research.

Response 8: Thank you for your expert advice. We have revised the conclusions section of the manuscript.

9. Little critical attention to the limitations of the study. For example: the authors did not mention whether the change in the VOC profile could have been a result of fruit maturity or microclimatic differences other than just crop structure.

Response 9: Thank you for this important observation. In order to reduce the errors caused by the differences in fruit maturity and microclimate. The samples used in this study were planted in the same area of the Zhejiang Citrus Research Institute. Peel samples were collected from randomly selected fruits with the same degree of maturity on 1 December. See lines 93-96 for an updated description.