Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comment:

The review provides a useful overview of volatile sulfur compounds (VSCs) in tomato plants, covering analytical methodologies, chemical diversity, sulfur metabolism, and potential biological functions. The topic is timely and relevant given the growing interest in plant volatilomics and sulfur-mediated stress responses. However, the manuscript remains largely descriptive and lacks sufficient critical analysis. Several sections summarize existing literature without adequately evaluating the strength of evidence, identifying knowledge gaps, or proposing future research directions. Therefore, substantial revision is recommended before publication.

Major Concerns

1. The manuscript states that HS-SPME-GC-MS may underestimate VSCs because of thermal degradation and poor sulfur selectivity. the review does not compare these methods (HS-SPME, DHS, and SBSE, TD-GC, GC-SCD, GC-PFPD, GC×GC-MS) with respect to detection limits, sulfur recovery, quantitative performance, and suitability for different classes of VSCs. A comparison table summarizing analytical performance would substantially strengthen the manuscript.
2. Figure 1 presents an integrated sulfur metabolism scheme. However, many of the proposed biosynthetic routes are inferred from studies in other plant species rather than experimentally demonstrated in tomato. The review frequently uses expressions such as: "may proceed", "is proposed" "is likely" ,which suggests substantial uncertainty.

The authors should more clearly indicate: which enzymes have been experimentally validated in tomato, which genes remain unidentified, which pathways are extrapolated from other species. Otherwise, eaders may overinterpret hypothetical pathways as established facts.

3. Although H₂S is an important sulfur-derived signaling molecule and a key intermediate in sulfur metabolism, a substantial portion of Section 5.2 is devoted to H₂S-mediated physiological responses. In contrast, the biological functions of other volatile sulfur compounds in tomato remain only briefly discussed. Given that the focus of this review is volatile sulfur compounds, a more balanced discussion of the available evidence for methanethiol, sulfides, thiols, and thiazoles would strengthen the manuscript.
4. While the review summarizes the current knowledge of VSCs in tomato plants, it does not clearly discuss the major challenges and future research priorities in this field. The addition of a short “Future Perspectives” section would strengthen the manuscript and help readers identify important directions for future studies.
5. Table 2 lists odor thresholds for many compounds but does not provide references for individual values. The source of these thresholds should be clarified.

Author Response

Comment: The review provides a useful overview of volatile sulfur compounds (VSCs) in tomato plants, covering analytical methodologies, chemical diversity, sulfur metabolism, and potential biological functions. The topic is timely and relevant given the growing interest in plant volatilomics and sulfur-mediated stress responses. However, the manuscript remains largely descriptive and lacks sufficient critical analysis. Several sections summarize existing literature without adequately evaluating the strength of evidence, identifying knowledge gaps, or proposing future research directions. Therefore, substantial revision is recommended before publication.

Response: We sincerely thank the Reviewer for the careful evaluation of our manuscript and for recognizing the relevance, timeliness, and usefulness of the topic. We are pleased that the Reviewer acknowledged the broad scope of the review, including analytical methodologies, chemical diversity, sulfur metabolism, and potential biological functions of VSCs in tomato plants. We also appreciate the critical comments regarding the descriptive character of some sections. In response, we have revised the manuscript to strengthen the critical analysis, more clearly identify current knowledge gaps, and better indicate future research directions, particularly in relation to targeted VSC analysis and sulfur-mediated stress responses.

Comment 1: The manuscript states that HS-SPME-GC-MS may underestimate VSCs because of thermal degradation and poor sulfur selectivity. the review does not compare these methods (HS-SPME, DHS, and SBSE, TD-GC, GC-SCD, GC-PFPD, GC×GC-MS) with respect to detection limits, sulfur recovery, quantitative performance, and suitability for different classes of VSCs. A comparison table summarizing analytical performance would substantially strengthen the manuscript.

Response 1: We thank the Reviewer for this valuable suggestion. We agree that a comparison of analytical approaches is important for understanding why the number of reported VSCs differs so strongly between studies. However, after carefully re-evaluating the tomato-specific literature, we found that a direct numerical comparison of detection limits, recovery, sulfur recovery, and quantitative performance is not possible in a reliable way, because most available studies were not validated specifically for VSCs and do not report comparable analytical parameters for individual sulfur compounds.

Therefore, instead of adding a potentially misleading table based on non-comparable data, we expanded the discussion following Table 1. We now explicitly discuss how detector type, extraction method, sorbent selectivity, sample preparation, tissue type, cultivar, developmental stage, and stress or post-harvest status may affect the number of identified VSCs. In particular, we emphasize that studies based on standard GC-MS or GC-MS/FID workflows usually reported only zero to two VSCs, whereas the study employing GC-PFPD identified seven VSCs, illustrating the practical advantage of sulfur-selective detection for this compound class. We also discuss the exceptional GC×GC-ToF-MS-based untargeted study reporting 58 VSCs and explain why such broad screening data should be interpreted cautiously without targeted confirmation using sulfur-selective detection, authentic standards, retention indices, and compound-specific validation.

We believe that this revised discussion more accurately reflects the current state of tomato VSC literature and avoids overinterpreting analytical performance parameters that are not consistently available across studies.

Comment 2: Figure 1 presents an integrated sulfur metabolism scheme. However, many of the proposed biosynthetic routes are inferred from studies in other plant species rather than experimentally demonstrated in tomato. The review frequently uses expressions such as: "may proceed", "is proposed" "is likely" ,which suggests substantial uncertainty.

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Comment 3: The authors should more clearly indicate: which enzymes have been experimentally validated in tomato, which genes remain unidentified, which pathways are extrapolated from other species. Otherwise, eaders may overinterpret hypothetical pathways as established facts.

Response 2 and 3: We would like to thank the Reviewer for this valuable and well-founded comments.  Our current understanding of sulfur metabolism, particularly VSC biosynthesis in tomato, is limited. In the case of tomatoes, knowledge is especially scarce, and even identifying several sulfur-containing volatile compounds has proven challenging due to their extremely low abundance and the associated analytical difficulties.

In response to this comment, we have thoroughly revised both Figure 1 and the corresponding section of the manuscript. Wherever possible, we now explicitly distinguish between mechanisms that have been demonstrated in tomato and those inferred from studies in other plant species. In the latter cases, we clearly indicate that these pathways are hypothetical and represent proposed mechanisms that may operate in tomato but have not yet been confirmed by experiments. We also attempted to include information on the regulation of S metabolism at enzymatic and molecular levels. However, as we highlight in the 'Future perspectives' section, the literature in this area is very limited.

Furthermore, we have redesigned Figure to highlight the hypothetical parts of the metabolic scheme more clearly, enabling readers to distinguish between pathways that have been experimentally supported and those that have been inferred. We believe that these revisions improve the accuracy and transparency of the manuscript, providing a more accurate reflection of the current state of knowledge in this field.

Comment 4: Although H₂S is an important sulfur-derived signaling molecule and a key intermediate in sulfur metabolism, a substantial portion of Section 5.2 is devoted to H₂S-mediated physiological responses. In contrast, the biological functions of other volatile sulfur compounds in tomato remain only briefly discussed. Given that the focus of this review is volatile sulfur compounds, a more balanced discussion of the available evidence for methanethiol, sulfides, thiols, and thiazoles would strengthen the manuscript.

Response 4: We sincerely thank the Reviewer for this valuable suggestion. We agree that the previous version of the manuscript placed considerable emphasis on H₂S-mediated physiological responses. This reflects the current state of knowledge, as H₂S is by far the best-studied sulfur-derived signaling molecule in plants, whereas information regarding the biological functions of other volatile sulfur compounds (VSCs) in tomato is extremely limited.

In response to the Reviewer's comment, we have revised Section 5.2 to achieve a better balance between H₂S and other classes of VSCs. We have shortened the discussion of H₂S-mediated physiological responses and tried to expand, as much as possible the sections describing the available evidence for the biological roles of methanethiol, sulfides, thiols, and thiazoles. Where direct evidence in tomato is lacking, we explicitly indicate whether the available information originates from studies in other plant species or from other biological systems, and we avoid overstating conclusions. We highlight in the 'Future perspectives' section, that the literature regarding methanethiol, sulfides, thiols, and thiazoles in tomato  is very limited.

Comment 5: While the review summarizes the current knowledge of VSCs in tomato plants, it does not clearly discuss the major challenges and future research priorities in this field. The addition of a short “Future Perspectives” section would strengthen the manuscript and help readers identify important directions for future studies.

Response 5: We sincerely thank the Reviewer for this valuable suggestion. We agree that highlighting the major knowledge gaps and future research priorities would improve the manuscript and provide readers with a broader perspective on the current state of the field.

In response to this comment, we have changed the section “Conclusions” to “Conclusions and future perspectives", in which we discuss the key challenges and promising directions for future research. In particular, we emphasize the limited understanding of the biosynthetic pathways and biological functions of many volatile sulfur compounds in tomato, the need for experimental validation of the proposed metabolic routes, the identification and functional characterization of enzymes involved in VSC biosynthesis, and the development of more sensitive analytical approaches for detecting these compounds. We also highlight the importance of investigating the roles of VSCs in plant physiology, stress responses, and plant–microbe interactions, as well as their potential applications in improving tomato  quality.

Comment 6: Table 2 lists odor thresholds for many compounds but does not provide references for individual values. The source of these thresholds should be clarified.

Response 6: The source of the odor threshold values was already indicated in the heading of Table 2; specifically, the data were taken from reference [44]: The Good Scents Company Information System. However, we agree that this information could be made clearer for the reader. To avoid any ambiguity, we have revised the table heading to explicitly state that the odor threshold values and aroma descriptions are based on The Good Scents Company Information System [44]. The reference has also been retained in the reference list.

Reviewer 2 Report

Comments and Suggestions for Authors

This review summarizes the analytical methods, chemical diversity, metabolic pathways, and biological functions (including plant defense and environmental adaptation) of volatile sulfur compounds (VSCs) in tomato plants. The topic is of both academic and applied value. However, the following issues still need to be addressed.
1. Sections 4 and 5 share exactly the same title, and the opening paragraph of Section 5 largely overlaps with content from Section 4. It is recommended to merge or reorganize these sections. Specifically, Section 4 should serve as a dedicated chapter on chemical classification, while Section 5 should focus on metabolism and biological functions.
2. Section 5.1 provides a rather basic description of sulfur metabolic pathways and lacks in-depth analysis of tomato specific regulatory mechanisms (e.g., particular gene families, transcription factors, tissue specific expression).
3. VSCs released from the rhizosphere (e.g., DMDS) play a distinct role in plant–microbe interactions, whereas VSCs in fruits mainly affect aroma quality. The metabolic regulation mechanisms underlying these two contexts are completely different. Moreover, wild tomatoes generally exhibit stronger stress tolerance, and their VSC profiles differ significantly from those of cultivated varieties. If possible, it is recommended to extend the discussion to include tissue specific and variety specific differences.
4. Much of the evidence regarding the role of VSCs in defense is inferred from other plants (e.g., Arabidopsis, cabbage) and lacks tomato specific experimental validation. It is advisable to clearly distinguish between “known” and “hypothesized” functions, and to highlight key hypotheses that require future verification.
5. Table 1 lists a wealth of studies. On this basis, it is recommended to add a discussion of the factors that cause the large variation in the number of identified VSCs across studies (e.g., extraction method, detector type, tomato variety, tissue type, etc.).
6. The speculation that “the presence of 2 methyl 3 furanthiol in fresh fruit may be related to overly harsh analytical conditions” is novel, but it should be supported by appropriate literature citations.

Author Response

Comment: This review summarizes the analytical methods, chemical diversity, metabolic pathways, and biological functions (including plant defense and environmental adaptation) of volatile sulfur compounds (VSCs) in tomato plants. The topic is of both academic and applied value. However, the following issues still need to be addressed.

Response: We sincerely thank the Reviewer for the careful evaluation of our manuscript and for recognizing the academic and applied relevance of the topic. We are pleased that the Reviewer acknowledged the broad scope of the review, including analytical methods, chemical diversity, metabolic pathways, and biological functions of volatile sulfur compounds in tomato plants. We have carefully considered all the issues raised by the Reviewer and revised the manuscript accordingly. Detailed responses to each comment are provided below.

Comment 1: 1. Sections 4 and 5 share exactly the same title, and the opening paragraph of Section 5 largely overlaps with content from Section 4. It is recommended to merge or reorganize these sections. Specifically, Section 4 should serve as a dedicated chapter on chemical classification, while Section 5 should focus on metabolism and biological functions.

Response 1: Thank you for this valuable suggestion. The titles of Sections 4 and 5 have now been amended. Section 4 is now titled “Classification and chemical diversity of VSCs in tomato”, and Section 5 is now titled “Metabolism and biological significance of VSCs in tomato”. We have revised the manuscript accordingly. Section 4 now focuses exclusively on the chemical classification of the compounds, while Section 5 has been revised to address their metabolism and biological functions. Additionally, overlapping introductory text has been shortened to improve the manuscript's overall structure and readability.

Comment 2: 2. Section 5.1 provides a rather basic description of sulfur metabolic pathways and lacks in-depth analysis of tomato specific regulatory mechanisms (e.g., particular gene families, transcription factors, tissue specific expression).

Reponse 2: We thank the Reviewer for this valuable comment and agree that the previous version of Section 5.1 lacked an in-depth analysis of the potential regulatory mechanisms underlying VSC biosynthesis in tomato plants, and a discussion of these mechanisms. However, the current literature provides limited information on which to base a comprehensive analysis of tomato-specific regulatory networks, including the involvement of particular gene families, transcription factors or tissue-specific expression patterns.

In the revised manuscript, we have expanded upon the molecular aspects of this section wherever possible and incorporated tomato-specific information where supported by the available evidence. Consequently, Section 5.1 has been substantially reorganised and revised to better reflect the current state of knowledge.

Furthermore, in the “Conclusions and Future Perspectives” section, we explicitly highlight existing knowledge gaps and emphasise the need for future studies to elucidate the molecular and regulatory mechanisms controlling VSC biosynthesis in tomato.

Comment 3: 3. VSCs released from the rhizosphere (e.g., DMDS) play a distinct role in plant–microbe interactions, whereas VSCs in fruits mainly affect aroma quality. The metabolic regulation mechanisms underlying these two contexts are completely different. Moreover, wild tomatoes generally exhibit stronger stress tolerance, and their VSC profiles differ significantly from those of cultivated varieties. If possible, it is recommended to extend the discussion to include tissue specific and variety specific differences.

Response 3: We fully agree with this statement, of course, and have tried to emphasise it in the current manuscript, particularly in the first paragraph of Section 5 andthe first paragraph of the Subsection 5.2. Unfortunately, a detailed discussion of the variability of VSC production and emissions in tomato plants, tissue and variety specific is impossible due to a lack of data. This issue is emphasised in the extensive 'Future Perspectives' section of the article.

Comment 4: 4. Much of the evidence regarding the role of VSCs in defense is inferred from other plants (e.g., Arabidopsis, cabbage) and lacks tomato specific experimental validation. It is advisable to clearly distinguish between “known” and “hypothesized” functions, and to highlight key hypotheses that require future verification.

Response 4: We thank the Reviewer for this important observation and agree that much of the current understanding of the role of VSCs in plant defense is derived from studies conducted in species other than tomato. In the revised manuscript, we have carefully distinguished between experimentally supported findings and hypotheses or conclusions extrapolated from other plant species. Wherever tomato-specific experimental evidence is lacking, we explicitly state this limitation and avoid presenting such mechanisms as established facts.

In addition, we have revised the relevant sections to clearly identify key hypotheses regarding the role of VSCs in tomato defense and, in the "Conclusions and Future Perspectives” section, we emphasize the need for future experimental studies to validate these proposed mechanisms in tomato.

Comment 5: 5. Table 1 lists a wealth of studies. On this basis, it is recommended to add a discussion of the factors that cause the large variation in the number of identified VSCs across studies (e.g., extraction method, detector type, tomato variety, tissue type, etc.).

Response 5: We agree that the large variation in the number of VSCs reported across studies required additional discussion. Therefore, we have expanded the relevant section discussing Table 1 to address the main factors responsible for these discrepancies, including detector type, extraction method, sorbent selectivity, sample preparation, tomato cultivar, tissue type, developmental stage, and stress or post-harvest status. We also clarified that studies based on standard GC-MS workflows may underestimate VSCs, whereas sulfur-selective detectors provide higher confidence for this compound class.

Comment 6: 6. The speculation that “the presence of 2 methyl 3 furanthiol in fresh fruit may be related to overly harsh analytical conditions” is novel, but it should be supported by appropriate literature citations.

Response 6: We thank the Reviewer for highlighting this inconsistency. Having carefully re-examined the relevant literature, we found that the available reports concerning the presence of 2-methyl-3-furanthiol in tomatoes are not entirely consistent. In light of this, we have revised the relevant section of the manuscript to ensure that it accurately reflects the current evidence and avoids unsupported speculation regarding tomato. Furthermore, prompted by this observation, we have thoroughly reviewed the entire manuscript to verify that all statements relating to tomatoes are precise and properly supported by the available literature. Where experimental evidence remains limited, we have ensured that these statements are appropriately worded.

Reviewer 3 Report

Comments and Suggestions for Authors

I have carefully read this review paper, which focuses on the state of the art of volatile organic sulfur compounds. I must say I find the work well written and structured, but I must say I find it difficult to find the novelty this research gives to the community. Section 2 has a description of the methodologies used for VCS analysis. Sections 3 and 4 are also very descriptive and I do not find they bring any novelty to the state of the art, let alone to be published in a high impact journal, like IJMS. 

Detailed Comments:

This work intends to make a summary of the current state of the art on VSC research, focusing on tomato crops. This research attempts to consolidate the current knowledge on VSC research;

However, in my view, this work brings little novelty considering already published reviews. This review does not add to the current state of the art of VSC, particularly if one considers the works of Bahatia (2024) https://doi.org/10.1016/j.comtox.2023.100295 or Giri et al (2014) https://doi.org/10.1016/j.procbio.2014.05.024

The authors could improve the manuscript by making substantial cuts to the current descriptive sections and adding new sections focusing on the future of VSC research aligned with the new generation of agriculture and ecological paradigms.

The references are adequate for the manuscript. The authors have produced really high-quality figures and should include more in the improved manuscript.

Author Response

Comment: I have carefully read this review paper, which focuses on the state of the art of volatile organic sulfur compounds. I must say I find the work well written and structured, but I must say I find it difficult to find the novelty this research gives to the community. Section 2 has a description of the methodologies used for VCS analysis. Sections 3 and 4 are also very descriptive and I do not find they bring any novelty to the state of the art, let alone to be published in a high impact journal, like IJMS. 

Response: We sincerely thank the Reviewer for the careful reading of our manuscript and for the positive assessment of its structure and writing. We understand the Reviewer’s concern regarding the novelty of the review, and we agree that this aspect should be stated more explicitly in the manuscript.

The novelty of this review does not lie in presenting a new analytical technique or a newly discovered metabolic pathway, but in integrating several issues that have so far been discussed separately. To the best of our knowledge, there is currently no review that combines: (i) the analytical limitations of VSC determination, especially their low abundance, reactivity, instability, thermolability and difficult quantification; (ii) the increasing biological and sensory importance attributed to sulfur-containing volatiles; and (iii) the specific relevance of these compounds in tomato plants. Previous studies and reviews have usually treated sulfur volatiles either as minor components of the general tomato volatilome, as aroma-active compounds in food products, or as individual metabolites linked to sulfur metabolism. In contrast, our review emphasizes that VSCs should not be considered only as a marginal fraction of untargeted volatilomic datasets, but as a chemically and biologically distinct group requiring dedicated analytical and biological investigation.

In particular, we aimed to highlight an important methodological gap: although interest in tomato VOCs has increased markedly, most studies still rely on broad untargeted metabolomic approaches, which are not optimized for VSCs. As a result, sulfur compounds are frequently underdetected, inconsistently reported, or not quantified with sufficient reliability. This is especially important because VSCs may occur at trace levels but still exert strong sensory and biological effects due to their low odor thresholds and high reactivity. Therefore, the review indicates the need for a more targeted research pathway focused specifically on sulfur volatiles, including appropriate extraction, detection and quantification strategies, rather than relying only on general untargeted VOC profiling.

Detailed Comments:

Comment: This work intends to make a summary of the current state of the art on VSC research, focusing on tomato crops. This research attempts to consolidate the current knowledge on VSC research;

However, in my view, this work brings little novelty considering already published reviews. This review does not add to the current state of the art of VSC, particularly if one considers the works of Bahatia (2024) https://doi.org/10.1016/j.comtox.2023.100295 or Giri et al (2014) https://doi.org/10.1016/j.procbio.2014.05.024

Response: We would like to thank you sincerely for submitting articles that have significantly broadened the scope of our own article. We consider the information you provided about computational approaches to characterising VSC chemical reactivity to be very valuable, as it enables a more comprehensive understanding of VSC behaviour, occurrence and transformation. We have therefore decided to include it in the introduction. We also included the work of Giri et al. (2014) in the article, enabling us to add to the manuscript the information that excess VSCs should be removed or neutralised. This information was absent from the previous version of the manuscript. We also considered the structure of both articles when revising our manuscript.

Comment: The authors could improve the manuscript by making substantial cuts to the current descriptive sections and adding new sections focusing on the future of VSC research aligned with the new generation of agriculture and ecological paradigms.

Response: We thank the Reviewer for this constructive suggestion. We agree that a stronger emphasis on future research directions would enhance the manuscript. In the revised version, we have substantially reorganized the manuscript, streamlined several descriptive sections, and expanded the “Conclusions and future perspectives” section. In particular, we discuss the major knowledge gaps and outline promising future research directions, including the need to elucidate the molecular mechanisms regulating VSC biosynthesis and function in tomato, as well as the potential application of this knowledge in sustainable crop production and next-generation agricultural strategies.

The references are adequate for the manuscript. The authors have produced really high-quality figures and should include more in the improved manuscript.

Response: We thank the Reviewer for the kind comment regarding the quality of the figures. In response to this suggestion, we have added a new figure illustrating the analytical methods used for the detection of VSCs (Figure 1).

Reviewer 4 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive and up-to-date overview of volatile sulphur compounds (VSCs) in tomatoes, detailing their biosynthesis, analytical challenges, and ecological significance.

Although it is very interesting, corrections are required before printing:

 

The heading 5. Classification and chemical diversity of VSCs in tomato. The exact same title has already been used as chapter 4.

 

The work requires intensive linguistic correction:

Page 1, Lines 36-37 , Lines 39-40

Page 2, Line 53, Line 64, Line 66, Lines 84-85

Page 3, Line 92,  Lines 116-117, Line 123

Page 4, Line 127, Lines 139-142, Line 152, Line 165, Lines 171-174

Page 5, Line 186 , Line 191

Page 6, Line 218, Line 227

Page 7, Line 245 , Line 255

Page 8, Line 308

Page 14, Line 521, Line 541, Line 560, Line 562

Author Response

Comment 1: This manuscript provides a comprehensive and up-to-date overview of volatile sulphur compounds (VSCs) in tomatoes, detailing their biosynthesis, analytical challenges, and ecological significance.
Although it is very interesting, corrections are required before printing:

Response 1: We sincerely thank the Reviewer for the careful reading of our manuscript and for the positive assessment of its comprehensive character and we are pleased that the Reviewer found the manuscript interesting.

 

Comment 2: The heading 5. Classification and chemical diversity of VSCs in tomato. The exact same title has already been used as chapter 4.

Response 2: Thank you for pointing out the error, we have changed the title of chapter 5.

 

Comment 3: The work requires intensive linguistic correction:

Page 1, Lines 36-37 , Lines 39-40

Page 2, Line 53, Line 64, Line 66, Lines 84-85

Page 3, Line 92,  Lines 116-117, Line 123

Page 4, Line 127, Lines 139-142, Line 152, Line 165, Lines 171-174

Page 5, Line 186 , Line 191

Page 6, Line 218, Line 227

Page 7, Line 245 , Line 255

Page 8, Line 308

Page 14, Line 521, Line 541, Line 560, Line 562

Response 3: We have carefully revised the manuscript and corrected the indicated language, grammar, and formatting issues. The revised version has also been thoroughly checked to improve clarity, readability, and consistency throughout the text.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have no further suggestions.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made changes to the manuscript, however my concerns about the limited novelty of the research remains unadressed.