Reactive Oxygen and Carbonyl Species: Dual Regulators of Abiotic Stress Signaling and Tolerance in Plants
, Shams Ur Rehman 2, Israt Jahan 3, Bir Jahangir Shirazy 4, Jotirmoy Chakrobortty 5, Md. Asadulla Al Galib 4, Rojina Akter 6, Sumaiya Farzana 7 and Yanjie Xie 8Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAbstract: It is simple, objective, and provides a clear justification for the importance of this literature review.
Keywords: Replace the two expressions highlighted in yellow in the attached file with other keywords. I suggest including "signaling molecules" and "plant stress resilience" as keywords.
Introduction: It is simple, objective, and provides a clear justification for the importance of this literature review.
The sequence: Topics 2.1 and 2.2 address the generation of ROS and RCS in plants in a simple and objective way. The figure presented is quite general and, in my opinion, could be a little more detailed regarding the generation of ROS and RCS; in any case, it is illustrative and helps to provide an overview of the two processes. Topics 3.1, 3.2, and 3.3 also address, in a simple, clear, and objective way, the responses to abiotic stresses, as well as the damage that can be caused by ROS and RCS when produced in higher concentrations in plants. Topic 4 is also well described and clearly elucidates the interaction between ROS and RCS. Regarding the figure, a suggestion for improvement was made, requiring greater detail in the internal processes that lead from ROS to the production of RCS and, from there, to the occurrence of the 4 final occurrence pathways indicated.
References: Although the topic is interesting and suggests the possibility of further study related to a better understanding of abiotic stress tolerance mechanisms in plants, most of the references used in the review are more than five years old. It would be beneficial for the authors of the study to verify if there are new published studies on the subject that could complement the review and/or update some of the information already described.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1
Abstract: It is simple, objective, and provides a clear justification for the importance of this literature review.
Responses: Thank you for the positive evaluation of the manuscript.
Keywords: Replace the two expressions highlighted in yellow in the attached file with other keywords. I suggest including "signaling molecules" and "plant stress resilience" as keywords.
Responses: According to the suggestions, we have added the "signaling molecules" and "plant stress resilience" as keywords in the revised manuscript.
Introduction: It is simple, objective, and provides a clear justification for the importance of this literature review.
Responses: Thank you for the positive evaluation of the manuscript.
The sequence: Topics 2.1 and 2.2 address the generation of ROS and RCS in plants in a simple and objective way. The figure presented is quite general and, in my opinion, could be a little more detailed regarding the generation of ROS and RCS; in any case, it is illustrative and helps to provide an overview of the two processes.
Responses: We thank the reviewer for the positive evaluation and constructive suggestion. In response, we have revised Figure 1 to include additional ROS-generating components, such as plasma membrane-bound NAD(P)H oxidase (RBOH) and apoplastic enzymes such as peroxidases. For RCS, we presented a simplified overview due to the complexity of their enzymatic and non-enzymatic generation. However, following the reviewer’s suggestion, we have improved Figure 2 by adding details on RCS formation from ROS and their dual roles in toxicity and signaling.
Topics 3.1, 3.2, and 3.3 also address, in a simple, clear, and objective way, the responses to abiotic stresses, as well as the damage that can be caused by ROS and RCS when produced in higher concentrations in plants. Topic 4 is also well described and clearly elucidates the interaction between ROS and RCS. Regarding the figure, a suggestion for improvement was made, requiring greater detail in the internal processes that lead from ROS to the production of RCS and, from there, to the occurrence of the 4 final occurrence pathways indicated.
Responses: We thank the reviewer for the positive evaluation and constructive suggestion. In Figure 2, the four arrows represent different physio-biochemical events illustrating the dual roles of ROS–RCS crosstalk, rather than distinct occurrence pathways. Therefore, we have revised Figure 2 by adding details on RCS formation from ROS and their dual roles in toxicity and signaling. To improve clarity of the crosstalk, the plasma membrane component has been removed, and the scheme has been simplified to show that abiotic stresses and phytohormone signaling induce ROS production, which subsequently leads to RCS generation. These RCS then integrate ROS-derived signals by modulating downstream signaling components, ultimately contributing to both cellular damage and adaptive signaling responses.
References: Although the topic is interesting and suggests the possibility of further study related to a better understanding of abiotic stress tolerance mechanisms in plants, most of the references used in the review are more than five years old. It would be beneficial for the authors of the study to verify if there are new published studies on the subject that could complement the review and/or update some of the information already described.
Responses: We thank the reviewer for this valuable comment regarding the inclusion of recent literature. We made every effort to incorporate the most relevant and impactful studies on ROS and RCS involvement in abiotic stress mechanisms. Although the number of newly published studies specifically addressing ROS–RCS interactions and abiotic stress mechanisms remains relatively limited, we have carefully re-examined the recent literature and updated the revised manuscript by incorporating available recent studies and adding relevant discussion to reflect current progress in this area. Here are following literature were added in the revised manuscript:
Van Veen et al. 2025; Pancheri et al. 2024; Swain et al. 2025; Zheng et al. 2025; Bao et al. 2025; Foyer and Hanke 2022; Lee and Kim 2024
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis m/s reviews the dual role of ROS and RCS in toxicity and signaling in plants. It is well known that ROS and RCS are both toxic, but also can participate in signalling. While this review does attempt to give a very broad overview of the literature, I feel the novelty of the m/s is rather low. There are so many good comprehensive reviews about ROS and other reactive molecules and their roles in signalling in plants, that it should clearly spelled out what is the novelty of this particular m/s. For example, it is difficult to see what the review offers over and above that of Zhou et al. J Plant Growth Regul 2022, 41, 119–142 (which is cited here).
Some important concepts are omitted. For example, I think the authors could have at least briefly discussed the concept of “retrograde” signalling in the chloroplast. The coordinated roles of ROS and RCS in retrograde signaling represent a sophisticated, two-tier system that allows chloroplasts to communicate with the nucleus and initiate protective responses. They function in a cascade, where ROS initiate the signal, and RCS both act as a secondary messenger and can serve as a signal on their own. There have been a number of reviews on this topic recently e.g.:
van Veen E, Küpers JJ, Gommers CMM. Plastids in a Pinch: Coordinating Stress and Developmental Responses Through Retrograde Signalling. Plant Cell Environ. 2025 48:6897-6911.
Pancheri T, Baur T, Roach T. Singlet-Oxygen-Mediated Regulation of Photosynthesis-Specific Genes: A Role for Reactive Electrophiles in Signal Transduction. Int J Mol Sci. 2024 Aug 2;25(15):8458. doi: 10.3390/ijms25158458.
Another concern is about quality of the figures. They are too simplified and do not indicate fully what is referred in the text. For example, in Figure 1 there is no indication of plasma membrane bound NAD(P)H oxidase, which produces superoxide in the apoplastic side of plasma membrane, and also no ROS producing cell wall enzymes such as peroxidases or aminooxidases are shown (although a brief mention in text about peroxidases exists). Figure 2 claims to illustrate cross-talk between ROS and RCS, however simple arrows from the apoplastic events to intracellular responses shown in the picture do no correspond to the concept of cross-talk.
Minor comments
Line 27 “leading” should be “leads”
Line 133 “contribute” should be “contributes”
Line 138 “homeostasis, where” better “homeostasis; in the peroxisomes”
Line 179 Here β-cyclocitral could also be listed.
Line 219ff. Important to state here that the primary role of the pathogen-induced oxidative burst is to inhibit / kill pathogens.
Line 267 delete “The”
Line 272 “Heavy metal stress”, better “Under heavy metal stress,”. I would suggest that this one sentence paragraph be “run-on” with the paragraph that follows.
Line 276 “In addition”, it’s not clear what the text that follows is in addition to. Maybe just delete?
Line 288 This section heading is a bit misleading, as most of the paragraphs are about toxic effects of RCS
Line 291 “Due to electrophilic nature” better “Due to their electrophilic nature”
Line 292 “optimal” better “at optimal”
Line 338 I think it should be noted here that ROS/RCS signalling is integrated with ALL other signalling systems in plants, e.g. sugars etc.
Line 349 Sentence starting “Abscisic acid… “ is very long, and the meaning cannot be understood, please re-word.
Line 392 “significant conceptual and translational challenges remain”, I cannot really understand this, what is meant by “translational challenges”?
Comments on the Quality of English LanguageAlthough he quality of English is acceptable and in general this m/s reads smoothly, in places the meaning is lost and text needs to be improved.
Author Response
Reviewer 2
This m/s reviews the dual role of ROS and RCS in toxicity and signaling in plants. It is well known that ROS and RCS are both toxic, but also can participate in signalling. While this review does attempt to give a very broad overview of the literature, I feel the novelty of the m/s is rather low. There are so many good comprehensive reviews about ROS and other reactive molecules and their roles in signalling in plants, that it should clearly spelled out what is the novelty of this particular m/s. For example, it is difficult to see what the review offers over and above that of Zhou et al. J Plant Growth Regul 2022, 41, 119–142 (which is cited here).
Responses: In contrast to Zhou et al. (2022), which provides a broad overview of multiple reactive species (ROS, RNS, RCS, and RSS) and their general crosstalk and applications, the present review specifically concentrates on the interaction between ROS and RCS. It uniquely emphasizes the hierarchical relationship in which ROS act as primary signals while RCS function as downstream mediators that refine and amplify signaling outputs. Moreover, this review develops a focused framework on their dual roles in toxicity and signaling under abiotic (salinity, drought, and heat) stress, rather than covering diverse reactive molecules and technological aspects. By narrowing the scope, it offers deeper insight into ROS–RCS integration and highlights specific research gaps relevant to stress tolerance mechanisms in plants. To clarify the novelty, we added the following sentences in the revised introduction:
“Although previous reviews have comprehensively discussed reactive species networks [22–24], often integrating ROS with other molecules such as RNS and RSS, a detailed mechanistic dissection of ROS–RCS interactions is still lacking. Moreover, while ROS signaling [22] and RCS toxicity [14] have been extensively examined separately, their integrated crosstalk and coordinated regulatory roles in plant stress responses have not been systematically resolved. In this review, we synthesize current knowledge on the dual roles of ROS and RCS as both damaging agents and indispensable signaling molecules in plant abiotic stress tolerance. We further highlight critical knowledge gaps and propose future research directions aimed at exploiting ROS–RCS-mediated redox net-works for improving crop resilience and advancing sustainable agricultural practices.”
Some important concepts are omitted. For example, I think the authors could have at least briefly discussed the concept of “retrograde” signalling in the chloroplast. The coordinated roles of ROS and RCS in retrograde signaling represent a sophisticated, two-tier system that allows chloroplasts to communicate with the nucleus and initiate protective responses. They function in a cascade, where ROS initiate the signal, and RCS both act as a secondary messenger and can serve as a signal on their own. There have been a number of reviews on this topic recently e.g.:
van Veen E, Küpers JJ, Gommers CMM. Plastids in a Pinch: Coordinating Stress and Developmental Responses Through Retrograde Signalling. Plant Cell Environ. 2025 48:6897-6911.
Pancheri T, Baur T, Roach T. Singlet-Oxygen-Mediated Regulation of Photosynthesis-Specific Genes: A Role for Reactive Electrophiles in Signal Transduction. Int J Mol Sci. 2024 Aug 2;25(15):8458. doi: 10.3390/ijms25158458.
Responses: Thank you for this insightful suggestion. We agree that retrograde signaling represents an important mechanistic framework linking ROS and RCS interactions. The recommended studies provide valuable mechanistic insights into retrograde signaling and have now been incorporated and cited in the revised manuscript. Accordingly, we have included a new paragraph in Section 4 (ROS–RCS crosstalk) to address this aspect.
“Beyond these cellular and tissue-level processes, ROS-RCS interactions also operate at the organellar scale to coordinate intracellular communication pathways. In particular, chloroplast-derived redox signals play a central role in mediating retrograde signaling to the nucleus under stress conditions [85,86]. In this context, plastid-derived ROS, particularly singlet oxygen (1O₂) and hydrogen peroxide (H₂O₂), act as primary retrograde signals that integrate environmental stress cues with developmental programs, enabling dynamic plastid–nucleus coordination [87]. These ROS further promote lipid peroxidation processes, leading to the generation of RCS, including electrophilic oxylipins, which function as secondary messengers and can also directly regulate nuclear gene expression [88]. Notably, singlet oxygen-mediated signaling has been shown to control photosynthesis-associated nuclear genes via RCS-dependent pathways, high-lighting a tightly coupled ROS–RCS signaling module [88]. Collectively, this ROS–RCS cascade constitutes a hierarchical, two-tier regulatory system that fine-tunes nuclear gene expression and facilitates rapid acclimation and stress adaptation in plants.”
Another concern is about quality of the figures. They are too simplified and do not indicate fully what is referred in the text. For example, in Figure 1 there is no indication of plasma membrane bound NAD(P)H oxidase, which produces superoxide in the apoplastic side of plasma membrane, and also no ROS producing cell wall enzymes such as peroxidases or amino-oxidases are shown (although a brief mention in text about peroxidases exists).
Responses: According to the comments, we have revised Figure 1 to include additional ROS-generating components, such as plasma membrane-bound NAD(P)H oxidase (RBOH) and apoplastic enzymes such as peroxidases.
Figure 2 claims to illustrate cross-talk between ROS and RCS, however simple arrows from the apoplastic events to intracellular responses shown in the picture do no correspond to the concept of cross-talk.
Responses: According to the comments, we have revised Figure 2 by adding details on RCS formation from ROS and their dual roles in toxicity and signaling. To improve clarity of the crosstalk, the plasma membrane component has been removed, and the scheme has been simplified to show that abiotic stresses and phytohormone signaling induce ROS production, which subsequently leads to RCS generation. These RCS then integrate ROS-derived signals by modulating downstream signaling components, ultimately contributing to both cellular damage and adaptive signaling responses.
Minor comments
Line 27 “leading” should be “leads”
Response: Accordingly, “leading” has been replaced by “leads” in the revised manuscript.
Line 133 “contribute” should be “contributes”
Response: Accordingly, “contribute” has been replaced by “contributes” in the revised manuscript.
Line 138 “homeostasis, where” better “homeostasis; in the peroxisomes”
Responses: According to the comments, we rewrote sentences as follows in the revised manuscript:
“In addition, peroxisomes are important contributors to cellular ROS homeostasis; in the peroxisomes, O₂ accepts electrons during various oxidative reactions.”
Line 179 Here β-cyclocitral could also be listed.
Responses: According to the comments, we have added the β-cyclocitral as a RCS in the revised manuscript.
Line 219ff. Important to state here that the primary role of the pathogen-induced oxidative burst is to inhibit / kill pathogens.
Responses: According to the comments, we added the following sentences in the revised manuscript:
“Importantly, during pathogen attack, the oxidative burst plays a direct antimicrobial role, contributing to the inhibition or killing of invading pathogens through the rapid accumulation of toxic ROS at the infection site. At the same time, pathogen-triggered ROS bursts are tightly controlled in duration and magnitude through the coordinated activation of NADPH oxidases and antioxidant systems, thereby minimizing collateral damage while also enabling effective defense signaling [49].”
Line 267 delete “The”
Responses: According to the comments, we have deleted “The” from the revised section.
Line 272 “Heavy metal stress”, better “Under heavy metal stress,”. I would suggest that this one sentence paragraph be “run-on” with the paragraph that follows.
Responses: According to the reviewer’s comments, “Heavy metal stress” has been revised to “Under heavy metal stress.” In addition, as suggested, this paragraph has been merged with the subsequent paragraph in the revised manuscript.
Line 276 “In addition”, it’s not clear what the text that follows is in addition to. Maybe just delete?
Responses: According to the comments, we have deleted “In addition” from the revised section.
Line 288 This section heading is a bit misleading, as most of the paragraphs are about toxic effects of RCS
Responses: Thank you for this insightful comment. We agree that the previous heading may not fully reflect the scope of the section, as it also discusses the toxic effects of RCS. To address this, we have revised the heading to better capture the dual role of RCS in signaling, toxicity, and abiotic stress responses. Here is the revised heading:
“Dual role of RCS in signaling, toxicity, and abiotic stress responses”
Line 291 “Due to electrophilic nature” better “Due to their electrophilic nature”
Responses: According to the comments, we have added “Due to their electrophilic nature” in the revised manuscript.
Line 292 “optimal” better “at optimal”
Responses: According to the comments, we revised the manuscript.
Line 338 I think it should be noted here that ROS/RCS signalling is integrated with ALL other signalling systems in plants, e.g. sugars etc.
Responses: Thank you for this valuable comment. We agree that ROS/RCS signaling does not operate in isolation but is intricately integrated with other signaling networks, including sugar, hormonal, and metabolic pathways. To address this, we have revised the sentence to reflect the broader signaling context. We revised the sentence as follows in the revised manuscript.
“ROS and RCS form an integrated signaling module that enables plants to translate rapid redox changes into sustained stress responses (Figure 2), which operates in close coordination with other signaling networks, including sugar, hormonal, and metabolic pathways [82].”
Line 349 Sentence starting “Abscisic acid… “ is very long, and the meaning cannot be understood, please re-word.
Responses: According to the comments, we have rewritten the sentences as follows in the revised manuscript:
“Abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid induce stomatal closure. These hormonal signals lead to the generation of ROS. The generated ROS then promote lipid peroxidation, resulting in the accumulation of RCS. RCS act downstream of ROS and upstream of Ca²⁺-dependent signaling events in guard cells”
Line 392 “significant conceptual and translational challenges remain”, I cannot really understand this, what is meant by “translational challenges”?
Responses: Thank you for this comment. By “translational challenges,” we refer to the difficulty of translating findings obtained under controlled laboratory conditions into practical applications under field conditions. While the roles of ROS and RCS are well characterized in model systems, their effective manipulation in crops is often constrained by environmental variability and complex field interactions. To improve clarity, we have revised the sentence accordingly.
“Despite substantial progress in elucidating the roles of ROS and RCS as central regulators of plant stress acclimation, significant conceptual and challenges in translating laboratory findings to field applications remain.”
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsAfter revision the text and figures were improved, although Figure 2 still does not illustrate cross-talk but a linear sequence of events. Authors made most of the changes according to my recommendations. However, the first sentence of the section 4 is still unclear, as if some word is missing.
Comments on the Quality of English LanguageEnglish is good.
