Genotype × Environment Shapes Fig Seed Oil Metabolic Fingerprinting

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Peer Review of " Genotype × Environment Shapes Fig Seed Oil Quality: Biochemical and FTIR‑ATR Fingerprinting"

Dear colleagues:

I have carefully read and studied your job. The author focused on the core scientific problem of G × E interaction, using 37 fig varieties, two years of field experiments, combined with biochemical analysis and FTIR-ATR spectroscopy, the data support is sufficient, and the research results have high reliability and practicability. The metabolic trade-off relationship between phenolics and lipid synthesis in fig seed oil was innovatively revealed, and the effectiveness of FTIR-ATR spectroscopy in capturing G × E-driven quality changes was verified, which provided a new perspective for quality control and rapid detection of oil crops. The discussion section closely combines the circular economy and industrial needs, and proposes the recommendation of stable high-value varieties. The practical guiding significance is clear, which is in line with the application orientation of food metabolomics research. If the author can effectively relieve my following concerns, I would be happy to recommend this manuscript to be published after min revision.

 

1.Some papers only simply listed ( such as ' extensive research has been conducted on the functional properties... [ 6-7-8-9-10-11-12 ] ' ), and did not integrate the core conclusions of these studies. The application status of FTIR-ATR spectroscopy in the study of G × E interaction is insufficiently described. Only 'reliable correlations between classical biochemical profiles and spectral fingerprints ' is mentioned, and the research gap of this technology in capturing environmental-driven quality changes is not explained.

Revised suggestion: ' extensive research has been conducted on the functional properties... [ 6-7-8-9-10-11-12 ] ' is rewritten as ' studies have confirmed that figs have functional properties such as anti-diabetes and anti-oxidation [6-12]. The active ingredients mainly depend on phenols and flavonoids in the fruit, but these studies focus more on the pulp of the fruit, and less on the seeds and their oils ' ; in addition, ' Although FTIR-ATR spectroscopy has been used for variety identification and quality control [ 15,28-31 ], there is no research to verify whether it can accurately capture the changes in the composition of fig seed oil caused by G × E interaction, which is difficult to meet the needs of cross-annual quality prediction ', which enhances the criticality of literature integration.

2. The gap of ' G × E interaction affects the quality of fig seed oil ' is not specific enough, and the specific quality indicators not involved in the existing research (such as phenols, flavonoids, synergistic changes in antioxidant activity and oil yield) are not explained; there is a lack of research on the key environmental factor of 'trans-annual environmental variation', and only' environmental conditions' are mentioned in general.

Suggestions for modification: 'systematic quantification of G × E interactions on fig seed oil geochemical remains absent ' was refined as ' no systematic study has been conducted to quantify the synergistic effects of G × E interactions on key quality indicators such as phenols, flavonoids content, antioxidant activity and oil yield in fig seed oil '. In addition, ' most of the existing studies are single-year single-point experiments, lacking the exploration of G × E interaction mechanism under inter-annual environmental variation, and cannot provide a basis for variety screening under different climatic conditions ', which enhances the specificity and persuasiveness of the gap.

3. The expression of ' sustainable valorization of this valuable agro-food and cosmetic resource ' in the research objectives is too broad and does not specify the specific application direction ; the hypothesis part does not clearly distinguish the logical level of the three hypotheses, and the expression is more lengthy.

Suggestions for modification : ' sustainable valorization... resource ' was modified to ' provide technical support for directional screening of fig seed oil varieties, cross-year quality prediction and stable application in food and cosmetics '. The three hypotheses were expressed in a simplified language : ( 1 ) Genotypic differences and cross-annual environmental variations jointly drive the changes in the biochemical characteristics of fig seed oil, and there is a metabolic trade-off between secondary metabolites such as phenols and flavonoids and oil synthesis ; ( 2 ) The composition change caused by G × E interaction can be captured by the characteristic peaks of FTIR-ATR spectra. ( 3 ) The combination of biochemical analysis and spectral technology can realize variety identification and cross-year quality prediction, and improve the clarity.

4.1 The selection of sample size in the material is lack of basis. The author did not explain the screening criteria of 37 genotypes ( such as whether based on genetic diversity analysis ), nor did he provide the efficacy analysis of sample size. Please amend the author.

4.2 The ATR correction parameters ( 45 ° incident angle, 1.5 refractive index ) of FTIR-ATR spectrum did not explain the selection basis, and did not mention whether to optimize the standard parameters based on pre-experiment or refer to the relevant literature.

5.Whether the experimental data of two years can effectively support the test results, at present, it generally needs 3-55 years of data analysis.

6. The conclusion part is too long. Generally speaking, only the core content of the work and the most important conclusions of the work are allowed to be displayed. Such as: ‘This study explored biochemical and spectral variability of fig seed oils from 37 Ficus carica L. genotypes (2023–2024), testing if genotypic differences and annual environmental conditions drive detectable biochemical shifts (including secondary metabolism-lipid biosynthesis trade-offs) via FTIR-ATR spectroscopy. Findings supported the hypothesis. Two-way ANOVA showed highly significant effects (p < 0.001) of Year, Variety, and their interaction. From 2023 to 2024, total phenolic content fell 36% (48.7→31.2 mg GAE/100g oil), DPPH activity rose 34% (43.7%→58.5%) (shift to lipophilic antioxidants under stress), and oil yield dropped (26.7%→21.2%), with a negative correlation (r = −0.49, p < 0.001) between yield and phenolics confirming metabolic competition. Cultivars ‘Dottato Perguerolles’, ‘VCR 276/49’, and ‘Condria’ showed stable high-value traits. FTIR-ATR captured G×E-driven variations at key peaks (3012, 2928, 1745, 1375, 1050 cm⁻¹) (ANOVA: F > 103, p < 0.001). Principal component analysis explained 75.4–84.5% of variance, enabling genotype/year discrimination and validating vibrational fingerprints for authentication. Future research should include Mediterranean multi-environment trials, targeted metabolomics/lipidomics, and transcriptomic/genome-wide studies to advance fig seed oil to precision breeding’ (I'm just giving you an example, and I'm not suggesting that you use it directly).

Comments on the Quality of English Language

Language polishing and revision seem to be necessary.

Author Response

Comment 1 : Some papers only simply listed ( such as ' extensive research has been conducted on the functional properties... [ 6-7-8-9-10-11-12 ] ' ), and did not integrate the core conclusions of these studies. The application status of FTIR-ATR spectroscopy in the study of G × E interaction is insufficiently described. Only 'reliable correlations between classical biochemical profiles and spectral fingerprints ' is mentioned, and the research gap of this technology in capturing environmental-driven quality changes is not explained.

Revised suggestion: ' extensive research has been conducted on the functional properties... [ 6-7-8-9-10-11-12 ] ' is rewritten as ' studies have confirmed that figs have functional properties such as anti-diabetes and anti-oxidation [6-12]. The active ingredients mainly depend on phenols and flavonoids in the fruit, but these studies focus more on the pulp of the fruit, and less on the seeds and their oils ' ; in addition, ' Although FTIR-ATR spectroscopy has been used for variety identification and quality control [ 15,28-31 ], there is no research to verify whether it can accurately capture the changes in the composition of fig seed oil caused by G × E interaction, which is difficult to meet the needs of cross-annual quality prediction ', which enhances the criticality of literature integration.

Response : Thank you for your insightful comments, which have strengthened the relevance of this introduction. We have incorporated them into the manuscript.

Comment 2 : The gap of ' G × E interaction affects the quality of fig seed oil ' is not specific enough, and the specific quality indicators not involved in the existing research (such as phenols, flavonoids, synergistic changes in antioxidant activity and oil yield) are not explained; there is a lack of research on the key environmental factor of 'trans-annual environmental variation', and only' environmental conditions' are mentioned in general.

Suggestions for modification : 'systematic quantification of G × E interactions on fig seed oil geochemical remains absent ' was refined as ' no systematic study has been conducted to quantify the synergistic effects of G × E interactions on key quality indicators such as phenols, flavonoids content, antioxidant activity and oil yield in fig seed oil '. In addition, ' most of the existing studies are single-year single-point experiments, lacking the exploration of G × E interaction mechanism under inter-annual environmental variation, and cannot provide a basis for variety screening under different climatic conditions ', which enhances the specificity and persuasiveness of the gap.

Response 2 : We sincerely thank you for these adjustments, which have further strengthened the scientific quality of this introduction

Comment 3 : The expression of ' sustainable valorization of this valuable agro-food and cosmetic resource ' in the research objectives is too broad and does not specify the specific application direction ; the hypothesis part does not clearly distinguish the logical level of the three hypotheses, and the expression is more lengthy.

Suggestions for modification : ' sustainable valorization... resource ' was modified to ' provide technical support for directional screening of fig seed oil varieties, cross-year quality prediction and stable application in food and cosmetics '. The three hypotheses were expressed in a simplified language : (1) Genotypic differences and cross-annual environmental variations jointly drive the changes in the biochemical characteristics of fig seed oil, and there is a metabolic trade-off between secondary metabolites such as phenols and flavonoids and oil synthesis ; (2) The composition change caused by G × E interaction can be captured by the characteristic peaks of FTIR-ATR spectra. (3) The combination of biochemical analysis and spectral technology can realize variety identification and cross-year quality prediction, and improve the clarity.

Response 3 : We thank you for these comments. We have revised this section by making concrete modifications in accordance with your suggestions.

Comment 4.1 :The selection of sample size in the material is lack of basis. The author did not explain the screening criteria of 37 genotypes (such as whether based on genetic diversity analysis), nor did he provide the efficacy analysis of sample size. Please amend the author.

Response 4.1 : The study's sample consists of 37 varieties selected from a large national fig collection maintained by INRA. This collection has been extensively characterized in previous molecular, morphometric, and metabolomic screenings by Khadari et al. (2005), Oukabli et al. (2010), Achtak (2010), and Hssaini et al. (2020–2023). This sample size is considered consistent for the specific objectives of this research: evaluating Genotype × Environment (GxE) effects on seed lipochemical profiles. Given that fig seeds are a high-value yet under-researched component of the fruit, this targeted selection ensures that our findings on seed valorization are both representative and scalable for industrial application.

Comment 4.2 : The ATR correction parameters (45 ° incident angle, 1.5 refractive index) of FTIR-ATR spectrum did not explain the selection basis and did not mention whether to optimize the standard parameters based on pre-experiment or refer to the relevant literature.

Response 4.2 : We thank the reviewer for this comment. The ATR correction parameters applied in this study (45° incident angle and refractive index of 1.5) were not arbitrarily selected but are based on well-established theoretical principles and widely accepted literature protocols for ATR-FTIR lipid analysis. The 45° incident angle corresponds to the fixed standard geometry of single-bounce ATR accessories, ensuring optimal total internal reflection and appropriate evanescent wave penetration, while the refractive index value of 1.5 is commonly reported for vegetable oils and triglyceride-based matrices. These parameters reflect intrinsic physical properties of the ATR system and lipid materials and are fully consistent with established reference methodologies and international guidelines for oil analysis using ATR-FTIR.

Comment 5 : Whether the experimental data of two years can effectively support the test results, at present, it generally needs 3-5 years of data analysis.

Response 5 : We sincerely thank the reviewer for this comment. Although studies over three to five years are ideal for fully analyzing genotype × environment interactions in perennial crops, our two-year study remains statistically robust and scientifically valid for our specific objectives. The evaluation of 37 genotypes over 2023–2024 provided a sufficient number of genotype-year combinations to detect significant G×E interactions while capturing substantial environmental variation, as shown by notable changes in TPC, DPPH, and oil yield. Our main objectives, namely identifying G×E patterns, detecting metabolic trade-offs, and developing rapid FTIR-ATR authentication protocols, can be achieved with a two-year follow-up, as supported by comparable studies on vegetable oils. Furthermore, we were able to identify stable, high-performing cultivars, providing actionable selection criteria, while acknowledging that these results form a foundation for ongoing multi-year validation studies in our breeding program.

Comment 6 : The conclusion part is too long. Generally speaking, only the core content of the work and the most important conclusions of the work are allowed to be displayed. Such as : ‘This study explored biochemical and spectral variability of fig seed oils from 37 Ficus carica L. genotypes (2023–2024), testing if genotypic differences and annual environmental conditions drive detectable biochemical shifts (including secondary metabolism-lipid biosynthesis trade-offs) via FTIR-ATR spectroscopy. Findings supported the hypothesis. Two-way ANOVA showed highly significant effects (p < 0.001) of Year, Variety, and their interaction. From 2023 to 2024, total phenolic content fell 36% (48.7→31.2 mg GAE/100g oil), DPPH activity rose 34% (43.7%→58.5%) (shift to lipophilic antioxidants under stress), and oil yield dropped (26.7%→21.2%), with a negative correlation (r = −0.49, p < 0.001) between yield and phenolics confirming metabolic competition. Cultivars ‘Dottato Perguerolles’, ‘VCR 276/49’, and ‘Condria’ showed stable high-value traits. FTIR-ATR captured G×E-driven variations at key peaks (3012, 2928, 1745, 1375, 1050 cm⁻¹) (ANOVA : F > 103, p < 0.001). Principal component analysis explained 75.4–84.5% of variance, enabling genotype/year discrimination and validating vibrational fingerprints for authentication. Future research should include Mediterranean multi-environment trials, targeted metabolomics/lipidomics, and transcriptomic/genome-wide studies to advance fig seed oil to precision breeding’ (I'm just giving you an example, and I'm not suggesting that you use it directly).

Response 6 : We sincerely thank you for this valuable comment. This section has been revised according to your suggestions in the manuscript.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Review for metabolites-4129159

General comments

This manuscript is interesting as it evaluates genotype × environment (G×E) effects on the biochemical characteristics of fig seed oil and explores FTIR-ATR fingerprinting. However, the central claim regarding “oil quality” is not sufficiently supported, because the study does not provide metabolite/chemical composition data that are commonly used to define oil quality (e.g., fatty acid profile, tocopherols, sterols, key minor compounds, etc.). The current results rely mainly on TPC/TFC and antioxidant assays, which makes the conclusions about “oil quality” potentially overstated.

To be suitable for publication, the authors should either (i) include additional analyses of metabolites/chemical markers that determine oil quality, or (ii) substantially revise the framing (title, aims, discussion, and conclusions) to accurately reflect what was measured.

Specific comments

1. Title
   The title remains too general and suggests a comprehensive assessment of “oil quality,” while the study does not determine key metabolites/markers of oil quality. The title should be revised to match the actual scope of the work, or the missing quality-related metabolite analyses should be added.

2. Abstract
   The abstract does not report all results mentioned in the methods (e.g., ABTS outcomes are not presented). In addition, the conclusions are still too broad and should be restricted to data-supported findings.

3. Introduction
   The introduction requires stronger scientific justification for measuring phenolics/antioxidant-related parameters in an oil matrix, as this is not always standard and needs clearer rationale (e.g., relevance of minor lipophilic compounds, extraction considerations, or the specific reason this approach is appropriate here).
   Moreover, the introduction currently emphasizes “oil quality,” while the study does not present the typical chemical composition analyses supporting that claim. This section should be revised for consistency with the methods and results.

4. Methods
   A key limitation is the absence of metabolite/chemical composition analyses that truly determine oil quality. If these analyses are added, the manuscript would be considerably strengthened and could be recommended for publication.
   For DPPH and ABTS, please use Trolox as a reference standard and report results as Trolox equivalents (TE)(e.g., TE per g sample, or another appropriate unit), which will improve comparability with the literature.

5. Results
   Please check Table 3 for consistency in numerical formatting (decimal comma vs decimal point). The manuscript should follow the journal’s formatting guidelines consistently.

6. Discussion
   Some paragraphs contain only a single sentence; these should be avoided. The discussion needs to be more comprehensive by linking parameters (e.g., oil yield vs TPC/TFC vs antioxidant activity vs FTIR-ATR patterns), relating findings to prior studies, and clearly explaining the novelty of this work.

7. Conclusions
   The conclusion should be more concise and should directly answer the stated objectives, focusing on the most important findings supported by the results.

8. References
   References must follow the journal guidelines and, where available, include DOIs. Please also ensure consistent formatting across the reference list.

Author Response

Comment 1 : The title remains too general and suggests a comprehensive assessment of “oil quality,” while the study does not determine key metabolites/markers of oil quality. The title should be revised to match the actual scope of the work, or the missing quality-related metabolite analyses should be added.

Response 1: We thank the reviewer for this comment. The title has been revised to better reflect the actual scope and objectives.

Comment 2 :The abstract does not report all results mentioned in the methods (e.g., ABTS outcomes are not presented). In addition, the conclusions are still too broad and should be restricted to data-supported findings.

Response 2 :Thank you for this insightful comment. These points have been incorporated into the abstract concerning the ABTTS results.

Comment 3. The introduction requires stronger scientific justification for measuring phenolics/antioxidant-related parameters in an oil matrix, as this is not always standard and needs clearer rationale (e.g., relevance of minor lipophilic compounds, extraction considerations, or the specific reason this approach is appropriate here).

Moreover, the introduction currently emphasizes “oil quality,” while the study does not present the typical chemical composition analyses supporting that claim. This section should be revised for consistency with the methods and results.

Response 3 : Thank you very much for your valuable comments. These have been addressed and the corresponding changes have been incorporated into the manuscript. We kindly invite you to review these modifications

Comment 4 : A key limitation is the absence of metabolite/chemical composition analyses that truly determine oil quality. If these analyses are added, the manuscript would be considerably strengthened and could be recommended for publication.

For DPPH and ABTS, please use Trolox as a reference standard and report results as Trolox equivalents (TE) (e.g., TE per g sample, or another appropriate unit), which will improve comparability with the literature.

Response 4 : We sincerely thank the reviewer for these constructive suggestions. Regarding chemical composition, our FTIR-ATR approach provides validated quantitative information on key oil quality parameters, complemented by our biochemical analyses (TPC, TFC, antioxidant capacity, oil yield) that reflect nutraceutical properties. We acknowledge that additional GC-MS analyses would provide further molecular detail and note this as a limitation and a perspective for future work. For the DPPH and ABTS assays, we evaluated the results as percentage inhibition, in line with similar published studies, which allows for reliable and consistent comparison of antioxidant capacities.

Comment 5 : Please check Table 3 for consistency in numerical formatting (decimal comma vs decimal point). The manuscript should follow the journal’s formatting guidelines consistently.

Response 5 : We sincerely thank you for this clear and valuable comment. These corrections have indeed been made in the manuscript.

Comment 6 :  Some paragraphs contain only a single sentence; these should be avoided. The discussion needs to be more comprehensive by linking parameters (e.g., oil yield vs TPC/TFC vs antioxidant activity vs FTIR-ATR patterns), relating findings to prior studies, and clearly explaining the novelty of this work.

Response 6 : We thank the reviewer for this comment. Our study provides a rigorous, integrated analysis combining biochemical measurements (oil content, total phenolics, total flavonoids, antioxidant activities) with FTIR-ATR spectral profiles, all supported by robust statistical analyses including ANOVA, PCA, boxplots, and heatmaps. This approach connects multiple key parameters and highlights genotype × environment effects across 37 fig cultivars over two consecutive years. By considering both intra-varietal and inter-annual variability, we reveal metabolic plasticity and the trade-off between lipid biosynthesis and phenolic accumulation, while FTIR-ATR fingerprints enable rapid variety discrimination and reliable cross-year quality prediction. The integration of these multi-parameter datasets, supported by rigorous statistical evaluation, constitutes the novelty of our work and demonstrates its relevance for identifying and selecting high-value nutraceutical cultivars.

Comment 7 : The conclusion should be more concise and should directly answer the stated objectives, focusing on the most important findings supported by the results.

Response 7 : We thank you for your valuable comment and have taken it into account by making the necessary revisions.

Comment 8 : References must follow the journal guidelines and, where available, include DOIs. Please also ensure consistent formatting across the reference list.

Response 8 : We thank you for these valuable comments. Indeed, the necessary modifications have been made to the reference list in accordance with the journal’s guidelines.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Accept in present form