Integrated Transcriptomics and Metabolomics Analyses Provide Insights into Heat Resistance in Passion Fruit (P. edulis f. flavicarpa)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The mss entitled "Integrated transcriptomics and metabolomics analyses provide insights into heat resistance in passion fruit" is a well written description of the comparison of two varieties of passion fruit differing in the tolerance to heat. The authors performed transcriptomic and metabolomic analyses, as well as physiological characterization of leaf biophysical traits, in response to heat stress.

Although the experiment only compared two varieties, with little known about their genetic origins and similarities, the authors were able to extract from these data the involvement of xanthine metabolism and, furthermore, show exogenous effects of the xanthine metabolism on seedling heat stress via transient gene expression studies.

The paper is worthy of publication in the special issue of Horticulturae, Fruit Tree Physiology.

A few minor comments for improvement.

1. The experiments are of 2 types, one on the young seedlings and the other on the developing fruit. It can be confusing to follow the differences in the results section and the two experimental systems with their results should be more clearly distinguished.
2. The weakness of comparing only two different varieties should be mentioned in the discussion regarding the conclusions that can be drawn. It would be especially useful if the research group could develop segregating F2 populations of these plants and make the correlations in seedlings of these. A longer term project but this mss provides the necessary background for future work.

Author Response

Comments 1: The experiments are of 2 types, one on the young seedlings and the other on the developing fruit. It can be confusing to follow the differences in the results section and the two experimental systems with their results should be more clearly distinguished.

Response 1: We appreciate the reviewer’s constructive feedback regarding the clarity of distinguishing the two experimental systems (seedling heat stress assay and field-grown mature plant sampling) in our study. We acknowledge that the presentation of results from these distinct systems could have been more explicitly separated to avoid confusion. First, we have reorganized the Results section to clearly separate findings from the two experimental systems under distinct subheadings: (2.1. Seedling Responses to Controlled Heat Stress; 2.2. Field-Grown Plant Responses to Natural High-Temperature Conditions). We explicitly state that the seedling experiments were designed to evaluate acute heat stress responses under controlled conditions, while the field experiments aimed to capture long-term adaptive differences between cultivars under natural high temperatures. This distinction aligns with our primary goal of comparing QM9 and F2 heat tolerance mechanisms. We added description to the Methods section (Sections 2.1 and 2.2) explaining the use of seedling experiments to determine differences in heat tolerance among passion fruit cultivars and field experiments to investigate differences in gene expression and metabolite accumulation among passion fruit cultivars under natural high temperature stress. All figures and tables clearly label whether data derive from "seedling experiments" or "field-grown plants", with consistent terminology throughout.

Comments 2: The weakness of comparing only two different varieties should be mentioned in the discussion regarding the conclusions that can be drawn. It would be especially useful if the research group could develop segregating F2 populations of these plants and make the correlations in seedlings of these. A longer term project but this mss provides the necessary background for future work.

Response 2: We agree that expanding the genetic diversity in follow-up work would strengthen the generalizability of our findings. We have added a paragraph in the Discussion section (Section 4) to acknowledge that comparison between only two cultivars may limit the broader applicability of our conclusions. Specifically, a limitation of this study is that the conclusions are drawn from comparison between only two passion fruit varieties (F2 and QM9). While this approach identified key metabolic and transcriptional differences associated with heat tolerance, the generalizability of these findings may be constrained by the limited genetic diversity represented. As suggested by the reviewers, we will investigate the utility of generating segregating F2 population from QM9 × F2 crosses in subsequent studies, which is a logical next step. Such population will facilitate QTL mapping or transcriptome association studies linking genotypic and phenotypic variation under heat stress. Our primary goal for this study is to highlight that the data presented here (e.g., candidate DEGs or pathways) lay the foundation for these future analyses. This study focuses on proof-of-concept differences between two well-characterized varieties under controlled and field conditions, prioritizing depth of mechanistic exploration (e.g., transcriptomics) over breadth of genetic diversity. This approach is consistent with our current goal to identify potential thermotolerance markers for validation in subsequent studies.

Reviewer 2 Report

Comments and Suggestions for Authors

The ms is very interesting and well written. It is suggested to write the whole file by using the third person instead the first one.

Several aspects should be considered in order to improve the ms; i.e. abstract should be rewritten to follow the typical structure.

The methodology and results sections in particular must be reviewed. Greater detail is expected in the methodology section so that the experiments can be repeated. It is unclear where the samples were taken, whether the trial was conducted in field or under controlled conditions, and the number of samples collected or how many transgenic events were obtained. Besides, the statistical analysis did not explain why some kind of samples were not considered. Although results were significant for research community, they should be presented accordingly to Horticulturae quality standards.

Besides, results must be showed in the order that methodology included the experiments. Most of the figures are almost impossible to correctly read them, please increase the resolution of figures or replace some of them by tables. In addition, various comments should be moved to discussion section.

Several comments were included along the text to be solved by authors.

Discussion must be included results of previous studies, especially in related genera since information for yellow passion fruit isn’t enough.

Comments for author File: Comments.pdf

Author Response

Comments 1: Several aspects should be considered in order to improve the ms; i.e. abstract should be rewritten to follow the typical structure.

Response 1: Thank you for the constructive comments. We have significantly revised the abstract to adhere to the standard structure (Introduction, Aim, Methods, Results, and Conclusions) while ensuring clarity and conciseness. See the revised Abstract section of the manuscript for details.

Comments 2: The methodology and results sections in particular must be reviewed. Greater detail is expected in the methodology section so that the experiments can be repeated. It is unclear where the samples were taken, whether the trial was conducted in field or under controlled conditions, and the number of samples collected or how many transgenic events were obtained. Besides, the statistical analysis did not explain why some kinds of samples were not considered. Although results were significant for research community, they should be presented accordingly to Horticulturae quality standards.

Response 2: We have carefully revised the Methods section to provide more detailed information, please see the Materials and Methods section for details. In response to the reviewer’s concern about the clarity of sample collection and experimental conditions, we have specified in Sections 2.1 and 2.2 the collection locations, tissue types, number of samples, and whether the experiments were conducted in the field or under controlled conditions, as well as the physiological differences between QM9 and F2 under heat stress and the overall purpose of the study. Regarding the reviewer’s comment on the number of transgenic events, we have clarified this in Section 2.8, noting that the transgenic events primarily involved silencing guanine deaminase (GDA) and xanthine dehydrogenase (XDH) genes using virus-induced gene silencing (VIGS). The reviewer also mentioned that the statistical analysis failed to account for the exclusion of some samples. After careful inspection, we note that Figure 1e does not include statistical analysis. We conducted two experimental replicates, but due to space limitations, only one replicate was shown. Considering some studies also present data from a single replicate (ref. 1), we initially followed this approach. However, we have now revised Figure 1e to include biological replicates. Although only two biological replicates were available, the revised figure sufficiently demonstrates the differences in DAB and NBT staining between QM9 and F2 leaves following heat stress.

Comments 3: Besides, results must be showed in the order that methodology included the experiments. Most of the figures are almost impossible to correctly read them, please increase the resolution of figures or replace some of them by tables. In addition, various comments should be moved to discussion section.

Response 3: We have modified the Methods section to match the order of the Results section and confirmed that the Results section now strictly follows the order described in the Methods section, making the manuscript more logical. All figures have been replaced with high-resolution versions (1200 ppi) to ensure clarity. We have moved all speculative or explanatory statements previously included in the Results section to the Discussion, reserving the Results for objective data presentation only. This separation aligns with journal standards and strengthens the manuscript’s structure.

Comments 4: Several comments were included along the text to be solved by authors.

Response 4: We have systematically addressed all specific comments and suggestions in the revised text by the reviewers and have clearly marked them in the revised manuscript through revisions and highlights.

Comments 5: Discussion must be included results of previous studies, especially in related genera since information for yellow passion fruit isn’t enough.

Response 5: We substantially revised the opening paragraph of the Discussion section by integrating previous studies on the physiological traits and partial molecular mechanisms underlying heat tolerance in passion fruit. Previous studies reported that certain passion fruit varieties develop a range of adaptive physiological responses under high-temperature stress, including modulation of antioxidant enzyme activity, osmotic regulation, and photosynthetic efficiency (ref. 2). Moreover, some studies have assessed and compared the temperature sensitivity of different passion fruit varieties (ref. 3 and 4).

 

The relevant modifications have been highlighted in yellow in the revised manuscript. Please refer to the updated version for further details.

 

References

1. Xiao, P., Qu, J., Wang, Y., Fang, T., Xiao, W., Wang, Y., Zhang, Y., Khan, M., Chen, Q., Xu, X., Li, C., Liu, J.H. Transcriptome and metabolome atlas reveals contributions of sphingosine and chlorogenic acid to cold tolerance in Citrus. Plant Physiol. 2024, 196, 634-650.
2. Wang, H., Zhao, J., Lai, M., Zhang, Y., Qiu, W., Li, Y., Tu, H., Ling, Q., Fu, X. Differential gene expression analysis and physiological response characteristics of passion fruit (Passiflora edulis) buds under high-temperature stress. Peer J. 2023, 11, e14839.
3. Shimada, A., Kubo, T., Tominaga, S., Yamamoto, M. Effect of temperature on photosynthesis characteristics in the passion fruits ‘summer queen’ and ‘ruby star’. Hort. J. 2017, 86, 194-199.
4. Matsuda, H., Ogata, T. Varietal differences in thermal response of passion fruit pollen germination. Trop. Agr. Develop. 2020, 64, 90-96.