Review Reports - The IAOx-Dependent IAA Biosynthesis Pathway: Acquired Insights, Paradigm Shifts, and Unresolved Questions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal

Plants (ISSN 2223-7747)

Manuscript ID plants-4013468

Type

Review

Title

The IAOx-Dependent IAA Biosynthesis Pathway: Established Understanding, Paradigm Shifts, and Future Challenges

Authors

Ming-Kun Ma , Verena Kriechbaumer * , Dong-Wei Di *

Section

Plant Molecular Biology

Special Issue

Advances in Plant Auxin Biology

Dear Editor

The text requires substantive and editorial revision.

Please find my comments below.

Yours sincerely,

Reviewer

Title

Please reconsider the title.

Abstract

Please expand on the meaning of each abbreviation used.

Please pay attention to the text of the abstract and make corrections in accordance with the following thematic structure according to the following points:

a) place the introduction in a broad context,
b) emphasize the purpose of the study,
c) briefly describe the main methods used,
d) summarize the main findings,
e) and indicate the main conclusions.

KEYWORDS

Please eliminate the terms appearing in the title of the manuscript and replace them with other phrases.

INTRODUCTION

32-58.

46-48. Please supplement the information on biosynthesis, provide details.

Please refer to the enzyme anthranilate synthase, which catalyzes the formation of anthranilic acid, a precursor in tryptophan biosynthesis.

59-99.

Please supplement the significance of the tryptamine (TAM) pathway in close relation to the topic of the manuscript.

Is Figure 1 entirely the work of the authors of the presented manuscript? If not, the consent of the authors of the chemical summary designs and diagrams is required.

Figure 1. If the authors prepared the diagrams themselves, please include them in the subsection – Materials and methods.

Please justify the relevance of the presented topic, indicate what has been done and what gaps exist in the literature.

10.

Please clearly and precisely formulate the purpose of the presented literature analysis in accordance with the topic of the manuscript.

32-99. When citing three to eight references instead of one or two general sentences, the reader expects substantive information about what other authors have achieved in the subject matter of the manuscript and in close connection with the topic of the manuscript. Please complete this information.

“[6-9], [12-14], [19-22], [23-28], [16,17,33], [6,18,34]”

12.

32-99 and the entire text of the manuscript and references – Latin names of genera and species should be written in italics (italics).

“Arabidopsis thaliana,” “Medicago truncatula,” etc.

13.

94. Please enter the correct citation.

Please enter a subchapter - Research materials and methods

In this subsection, please provide the following information:

- which literature search databases were used

- what period of time the literature analysis covers and why, justify

- what keywords were used to search the literature

- what scheme was adopted for the analysis of specific literature items

- what was taken into account when selecting the cited literature items, etc.

114-245.

15.

The text is heavy, the authors accept information uncritically. I suggest adding other thematic sub-points corresponding to the topic of the manuscript, because for this type of publication, the two thematic sub-points presented, in my opinion, provide too little substantive information on such a well-known and extensive topic.

In each sub-section, please insert a diagram, table, or graph as an illustrative representation of the subject matter of the sub-section. This will significantly improve the quality of the manuscript and reduce the heaviness of the text. 16.

The entire manuscript contains approximately 3,862 words (excluding affiliations, additional information such as acknowledgments, etc., and references), including line numbers.

Introduction: approximately 771, or about 20%

Subchapters 2 and 3: 1,646, or about 43%

Future horizons: 959, or about 25%

Concluding Perspective = 486, which is approximately 12%

Please maintain the appropriate proportion in the length of the subsections in accordance with the accepted rules for this type of manuscript.

17.

Please make a correction to the name of the subsection; I suggest “Future research perspectives” instead of “Future horizons.”

18.

Please eliminate the citation of literature in the “Future horizons” subchapter, as this is the creative opinion of the authors writing the manuscript. Move the citations of literature to the appropriate subchapters.

Please use the term “Conclusions” instead of “Concluding Perspective”; do not cite literature in this place.

22.

Please check each reference step by step and make corrections in accordance with the guidelines for authors, e.g., references 12-14, 16, 19, 23, 26, 28, 31, 34, 38, 42, etc.

Author Response

Reviewer 1：

1) Please reconsider the title.

Done as suggested. The new title has been revised as follows: “The IAOx-Dependent IAA Biosynthesis Pathway: Acquired Insights, Paradigm Shifts, and Unresolved Questions”

2) Please expand on the meaning of each abbreviation used.

Done as suggested.

3) Please pay attention to the text of the abstract and make corrections in accordance with the following thematic structure according to the following points:

a) place the introduction in a broad context,
b) emphasize the purpose of the study,
c) briefly describe the main methods used,
d) summarize the main findings,
e) and indicate the main conclusions.

Thank you very much for the clear and professional structural guidance for revising the abstract. We fully agree to rewrite the abstract according to the suggested framework: “The auxin indole-3-acetic acid (IAA) is essential for plant growth and stress adaptation. Its biosynthesis via the indole-3-acetaldoxime (IAOx) pathway has recently undergone a paradigm shift. Recent genetic and metabolomic studies have fundamentally revised the indole-3-acetaldoxime (IAOx) pathway from a linear route (IAOx→IAN→IAM→IAA) to a dynamic network. This review synthesizes this paradigm shift by integrating evidence from key Arabidopsis studies. Crucially, mutants disrupting multiple downstream enzyme families fail to block IAA overproduction in the IAOx-accumulating superroot 2 (sur2) background. Functioning as a central branching point between auxin and defense metabolism, the tryptophan-derived metabolite IAOx, along with indole-3-acetonitrile (IAN) and indole-3-acetamide (IAM), elicits auxin responses via independent, tissue-specific pathways, with no metabolic requirement for IAM as a universal intermediate. Furthermore, IAN and IAM levels do not increase with massive IAOx accumulation, indicating a bypass route from IAOx to IAA. We conclude that IAOx acts as a central metabolic hub, partitioning flux competitively between growth and defense. Resolving the unknown IAOx-converting enzyme, the signaling roles of IAN/IAM, and the logic of metabolic channeling is vital to understanding how plants integrate hormonal and stress responses”

4) KEYWORDS: Please eliminate the terms appearing in the title of the manuscript and replace them with other phrases.

Done as suggested. The new keywords have been revised as follows: “Keywords: auxin biosynthesis, indole-3-acetaldoxime indole-3-acetamide, indole-3-acetonitrile, growth-defense trade-off”

5) INTRODUCTION 32-58. Please supplement the information on biosynthesis, provide details.

Done as suggested. The revised contents described as follows:” IAA biosynthesis serves as the foundation of this regulatory network, originating from chorismate in the chloroplast, which is … IGP is predominantly channeled to Trp through the tryptophan synthase complex (TSA/TSB) for entry into TD pathways, while also serving as the substrate for indole synthesis via indole synthase (INS) to initiate the TI route [16,17].”

6) Please refer to the enzyme anthranilate synthase, which catalyzes the formation of anthranilic acid, a precursor in tryptophan biosynthesis.

Thank you for this correction. We agree that explicitly mentioning "anthranilate synthase (AS)" will make the description of this biochemical step more complete and clearer. We have revised the text at the following location: “During Trp synthesis in Arabidopsis thaliana, chorismate is converted to anthranilate by the anthranilate synthase (AS). This rate-limiting step is mediated by the WEI2/ASA1 and WEI7/ASB1 genes, encoding the α and β subunits of the AS enzyme, respectively [12–14].”

7) 59-99, Please supplement the significance of the tryptamine (TAM) pathway in close relation to the topic of the manuscript.

In accordance with your suggestion, we have supplemented the detailed description of the tryptamine (TAM) pathway in the relevant section. The specific additions are as follows: “Although the tryptamine (TAM) pathway has been proposed, its physiological significance remains controversial, with potential functionality restricted to specific tissues or particular conditions [32]. TAM exhibits auxin-like activity, but several lines of evidence challenge its role as a major IAA precursor: it is often found at high, non-specific levels across species, originates from distinct Trp pools, and its proposed conversion to IAA lacks robust enzymatic support [33-35]. While tissue-specific conversion (e.g., in pea roots) suggests limited, context-dependent functionality, TAM is not considered a conserved, primary route for IAA biosynthesis.”

8) Is Figure 1 entirely the work of the authors of the presented manuscript? If not, the consent of the authors of the chemical summary designs and diagrams is required.

Thank you for your rigorous inquiry regarding the copyright of Figure 1. We provide the following clarification: Figure 1 is designed to visually summarize the paradigm shift in the IAOx pathway from the linear model to the network model. This figure was created entirely originally by the authors of this manuscript, based on well-established scientific knowledge in the field. In addition, we also added the following statement to the Figure Legends, which is described as follows: “All depicted biochemical pathways and molecular structures were drawn based on established knowledge from the cited literature.”

9) Figure 1. If the authors prepared the diagrams themselves, please include them in the subsection – Materials and methods.

Thank you for highlighting this technical point. Figure 1 ("Paradigm shift in the IAOx-dependent auxin biosynthesis pathway: An evolving view") is indeed an original diagram created specifically for this review by the authors. In accordance with your suggestion, we will add the following statement to the Figure Legends, which is described as follows: “All depicted biochemical pathways and molecular structures were drawn based on established knowledge from the cited literature.”

Thank you again for your meticulous review.

10) Please justify the relevance of the presented topic, indicate what has been done and what gaps exist in the literature.

Thank you for this important comment, which prompts us to further clarify and highlight the rationale of our review.

The three core elements you mentioned, the relevance of the topic, what has been accomplished, and what gaps exist, are indeed crucial components of an introduction. In our current manuscript, this information is present but distributed across several paragraphs of the Introduction:

Topic relevance: The opening paragraphs establish the central importance of IAA, and subsequent text highlights the unique position of the IAOx pathway as a critical node linking growth and defense metabolism.

Accomplishments: Sections 2 (The established paradigm) and 3 (Contemporary revisions) systematically cover the field's progression from the establishment of the linear model to its recent fundamental revision by work such as Fenech et al. (2025).

Existing gaps: Section 4 (Future research perspectives) is dedicated to identifying and discussing current knowledge boundaries and key unresolved questions (e.g., the unknown enzyme, re-evaluation of IAN/IAM functions).

To directly address your point and make this logical framework immediately clear to the reader, we have revised the end of Introduction, which is described as follows: “The IAOx pathway has recently undergone significant conceptual revisions, with transformative insights from seminal work by Fenech et al. (2025) compelling comprehensive re-evaluation of its metabolic fluxes …It should be noted that the mechanistic insights and paradigm shifts discussed herein derive primarily from studies in Brassicaceae species, notably Arabidopsis.”

We believe that incorporating this transitional paragraph will significantly strengthen the manuscript's logical structure, clearly presenting to the reader why this topic is reviewed, what we currently know, and what we still need to learn, thereby perfectly setting the stage for the stated aims and scope of this review.

11) Please clearly and precisely formulate the purpose of the presented literature analysis in accordance with the topic of the manuscript.

Thank you for your comment regarding the purpose of the presented literature analysis. The aim of this review is clearly stated toward the end of the Abstract and at the conclusion of the Introduction section.

Specifically, in the Introduction, we add the following contents : “Against this backdrop of renewed interest and paradigm evolution, this review systematically traces the research trajectory of the IAOx pathway, providing an overview of established conceptual frameworks, a critical analysis of emerging evidence, and an identification of pivotal scientific questions demanding future investigation.”

We believe this statement clearly formulates the core purposes of our literature analysis as:

1) To systematically trace the research trajectory of the IAOx-dependent IAA biosynthesis pathway.

2) To critically analyze the paradigm shift in this field from the "linear model" to the "network model."

3) To identify and propose key questions and future directions for the field.

12) 32-99. When citing three to eight references instead of one or two general sentences, the reader expects substantive information about what other authors have achieved in the subject matter of the manuscript and in close connection with the topic of the manuscript. Please complete this information.“[6-9], [12-14], [19-22], [23-28], [16,17,33], [6,18,34]”

Thank you for raising the important point regarding the informational content of literature citations. We fully understand and appreciate your concern that providing sufficient substantive background when citing multiple references enhances the clarity and persuasiveness of the argument.

In writing this review, our primary consideration was to maintain a clear and fluent narrative centered on the “paradigm shift.” Therefore, for concepts that are well-established and considered foundational background knowledge in the field (e.g., the general framework of auxin homeostasis regulation summarized by [6-9], or the basic function of the TAA1/TAR aminotransferase family represented by [19–22]), we opted for concise citations to authoritative sources to avoid interrupting the flow of the core logic with excessive detail.

Nevertheless, upon careful consideration of your comment, we agree that providing more specific information at certain key junctures that define major pathways or involve core evidence for the recent paradigm shift would be beneficial. This would allow readers to better grasp the key research advances underpinning our arguments.

Accordingly, we will implement targeted enhancements to the following citation groups you noted, which are crucial for understanding the "paradigm shift":

1) For [23–28] (YUCCA family):“followed by oxidative conversion of IPyA to IAA catalyzed by YUCCA (YUC) family flavin monooxygenases, a reaction recognized as the rate-limiting step [23–28]. The identification of the YUC gene family [27], coupled with evidence of its members’ functional redundancy, distinct spatiotemporal expression patterns [18, 24, 26], and functional coordination with the upstream TAA1/TAR enzymes [23,28] collectively underscores the centrality of the IPyA pathway in plant auxin biosynthesis.”

2) For [16,17,33] (Tryptophan-independent pathway): “The TI pathway … ultimately yielding IAA [16,17,33]. These studies, using genetic and isotopic tracing evidence, suggested a potential role for this pathway in embryogenesis [17] and maintaining basal IAA levels [16], although its precise enzymatic mechanisms remain elusive to date [33].”

3) For [6,18,34] (Multi-layered regulatory network):“… to maintain IAA dynamic balance and support plant growth and environmental adaptation [6,18,34]. This involves integrated control at multiple levels, from epigenetic and transcriptional regulation [6,34] to the precise spatiotemporal control of key biosynthetic genes like YUC [18].”

For the remaining citation groups (e.g., [12-14]), which describe very specific, textbook-level steps in tryptophan synthesis, we believe maintaining citation conciseness best serves the narrative flow in the current context. However, if you consider any of these groups to be of particular importance for your review, we are certainly willing to add clarifying information based on your further guidance.

We believe that this focused enhancement effectively addresses your request for greater depth of information without compromising the clarity of the manuscript's central storyline.

Thank you again for your insightful review.

13) 32-99 and the entire text of the manuscript and references – Latin names of genera and species should be written in italics (italics).

“Arabidopsis thaliana,” “Medicago truncatula,” etc.

Done as suggested.

14) 94. Please enter the correct citation.

Done as suggested

15) Please enter a subchapter - Research materials and methods

In this subsection, please provide the following information:

- which literature search databases were used

- what period of time the literature analysis covers and why, justify

- what keywords were used to search the literature

- what scheme was adopted for the analysis of specific literature items

- what was taken into account when selecting the cited literature items, etc.

Thank you for your attention to the rigor of the literature selection in our manuscript. We appreciate and understand the rationale behind your request.

Firstly, we would like to clarify that this manuscript is a Review article, whose standard format focuses on the synthesis, critique, and prospect of existing knowledge, and typically does not include a dedicated "Materials and Methods" section akin to original research articles. Incorporating detailed search strategies into the main text could disrupt the narrative flow, which is centered on the core theme of "paradigm shift."

However, to fully address your concern for methodological transparency and to demonstrate that this review is built upon a systematic and rigorous literature analysis, we are pleased to provide you directly with all the requested information here for your perusal during the review process. These details attest to the systematic and reproducible nature of our work:

1) Search databases: The literature search for this review was conducted primarily using PubMed, Web of Science, and Google Scholar to ensure comprehensive coverage of core journals and significant preprints in the life sciences.

2) Timeframe and justification: The search was limited to publications from the key early studies of the pathway (circa 2000, e.g., identification of CYP79Bs) up to June 2025. This range was chosen to encompass the entire history of conceptual evolution of the IAOx pathway, from its proposal and the establishment of the linear model to its recent comprehensive revision by work such as that of Fenech et al. (2025).

3) Keyword strategy: We employed combined keyword searches, including core terms and their variants: "indole-3-acetaldoxime" OR "IAOx", "auxin biosynthesis", "CYP79B" OR "CYP83B1" OR "sur2", "Arabidopsis", "Brassicaceae", supplemented by related terms such as "indole-3-acetonitrile" (IAN), "indole-3-acetamide" (IAM), "camalexin", and "paradigm shift" to capture different research facets.

4) Scheme for literature analysis: For the retrieved literature, we adopted the following analytical framework: Identifying key papers that established the IAOx linear model (e.g., Sugawara et al., 2009); Systematically locating studies reporting genetic or biochemical evidence inconsistent with the linear model, serving as harbingers of the paradigm shift; Critically analyzing the core study (especially Fenech et al., 2025) that directly led to the model's reconceptualization; Comparing old and new evidence to synthesize current knowledge boundaries and define key future research questions.

5) Criteria for selecting cited literature: In deciding which literature to cite, we primarily considered: Direct scientific relevance, quality of evidence and impact, and

contribution to the narrative.

We hope this detailed supplementary account adequately demonstrates that the literature foundation of this review is comprehensive, systematic, and critically curated. We remain convinced that maintaining a focused and fluent narrative in the main text best serves to communicate the exciting, fundamental progress in this field to our readers.

Thank you again for your time and valuable insights.

16) The text is heavy, the authors accept information uncritically. I suggest adding other thematic sub-points corresponding to the topic of the manuscript, because for this type of publication, the two thematic sub-points presented, in my opinion, provide too little substantive information on such a well-known and extensive topic.

Thank you for raising this point, which prompted us to deeply reflect on the critical depth and informational value of our manuscript. We fully agree that a review on such a foundational topic should offer analytical perspectives and substantial insights beyond mere description.

In direct response to your concerns, we have implemented a substantial and structural enhancement in the revised manuscript, most prominently through a significant expansion and reorganization of the "Future Research Perspectives" section (Section 4). This revision directly addresses your call for "more substantive information" and markedly elevates the critical discourse:

1) We have reorganized the future challenges into five interconnected core scientific questions (e.g., identifying and regulating the unknown enzyme; re-evaluating IAN/IAM as signals; the cellular logic of metabolic channeling; evolutionary trajectory; network integration under stress). This systematic breakdown itself embodies critical thinking by defining the boundaries of current knowledge and mapping pathways to address them.

2) Within each future direction, we move beyond listing questions to engage in in-depth, evidence-informed speculation grounded in the evidence reviewed in Sections 2 & 3. For instance, regarding the "unknown enzyme," we propose and critically evaluate several plausible biochemical mechanisms (novel enzyme, metabolon, compartmentalization), analyzing their logical underpinnings and experimental challenges. This transforms the outlook from a wish list into a theoretical framework guiding future experimental design.

3) We have carefully considered the suggestion to add other thematic sub-points. We believe the manuscript's narrative power lies in its clear, single-threaded focus on tracing the coherent trajectory of the paradigm shift from the "linear model" to the "dynamic network." Concentrating on elucidating the evidence for this shift (Sections 2 & 3) and the entirely new landscape of questions it reveals (Section 4) best highlights the field's fundamental progress. The newly added, highly integrated future perspectives section has already greatly enriched the substantive content and analytical depth along this central storyline.

We are confident that the revised manuscript, through this focused yet in-depth approach, has effectively enhanced its critical stance and now provides core readers with significantly increased, insightful information on the field's current state and future. We sincerely appreciate your role in prompting this important improvement.

17) In each sub-section, please insert a diagram, table, or graph as an illustrative representation of the subject matter of the sub-section. This will significantly improve the quality of the manuscript and reduce the heaviness of the text.

The entire manuscript contains approximately 3,862 words (excluding affiliations, additional information such as acknowledgments, etc., and references), including line numbers.

Introduction: approximately 771, or about 20%

Subchapters 2 and 3: 1,646, or about 43%

Future horizons: 959, or about 25%

Concluding Perspective = 486, which is approximately 12%

Please maintain the appropriate proportion in the length of the subsections in accordance with the accepted rules for this type of manuscript.

Thank you for your meticulous and constructive feedback. We fully agree that an appropriate balance in section length helps readers grasp the key points. We have noted the word count distribution. The manuscript's focus is indeed on elucidating the field's "paradigm shift" (Sections 2 & 3) and the resulting "future directions" (Section 4), with the Introduction and Conclusion serving to frame these core sections. Following the integration of the Figures above, we will correspondingly refine and streamline the text.

We believe these revisions will effectively reduce textual density and substantially improve the manuscript's visual appeal and argumentative clarity.

Thank you again for your valuable time and expert guidance.

18) Please make a correction to the name of the subsection; I suggest “Future research perspectives” instead of “Future horizons.”

Done as suggested.

19) Please eliminate the citation of literature in the “Future horizons” subchapter, as this is the creative opinion of the authors writing the manuscript. Move the citations of literature to the appropriate subchapters.

Thank you for your valuable comment. We fully agree that the "Future horizons" section should focus on presenting the authors' creative perspectives and research proposals based on existing evidence, rather than citing literature to substantiate these forward-looking viewpoints.

In accordance with your suggestion, we have removed all literature citations from every subsection (i-v) of Section 4, "Future horizons" (e.g., [36], [54], [30], [35], [40], [41], [39], [68], etc.) and reintegrated these removed citations into the appropriate earlier subsections where the established facts they support are described.

20) Please use the term “Conclusions” instead of “Concluding Perspective”; do not cite literature in this place.

Thank you for your valuable suggestion. In accordance with your recommendation, we have revised the section title from "Concluding Perspective" to "Conclusion" and have integrated the content containing cited references into the Introduction. The remaining text has been reorganized and revised accordingly. The specific modifications are detailed below: “5. Conclusions The IAOx-dependent IAA biosynthesis pathway, once viewed as a linear metabolic route, has now emerged as a dynamic, channeled network that bridges auxin production with defense metabolism. The paradigm shift initiated by recent genetic and metabolomic studies not only redefines the role of IAOx, IAN, and IAM but also reveals a sophisticated regulatory logic governing resource allocation between growth and defense programs.

Looking forward, several frontier challenges remain. Future research must prioritize the identification of the unknown enzyme(s) catalyzing IAOx-to-IAA conversion, elucidate the tissue-specific signaling functions of IAN and IAM, and unravel the mechanisms of metabolic channeling under environmental stress. Moreover, integrating the IAOx pathway into the broader auxin signaling network—encompassing both nuclear and cell-surface signaling modules—will be essential to understand how plants dynamically balance growth and defense in fluctuating environments. Advancing these questions will require interdisciplinary approaches, including single-cell omics, spatial metabolomics, and systems biology modeling, to move from a descriptive framework toward a predictive understanding of auxin-driven plant adaptation.”

21) Please check each reference step by step and make corrections in accordance with the guidelines for authors, e.g., references 12-14, 16, 19, 23, 26, 28, 31, 34, 38, 42, etc.

Thank you for the helpful suggestion. In accordance with the “Guidelines for Authors,” we have meticulously reviewed all references cited in the manuscript to ensure consistency and accuracy in formatting.

Reviewer 2 Report

Comments and Suggestions for Authors

Write the scientific names (genus and species) in italics, for example: Arabidopsis thaliana, Medicago truncatula, etc.

In Figure 1. Paradigm shift in the IAOx-dependent auxin biosynthesis pathway: An evolving view. 1) The chemical formula for indole-3-acetonitrile (IAN) is incorrect; its molecular structure corresponds to that of indole-3-acetic acid (IAA). Correct the chemical structure. 2) Part "A" of Figure 1 is equal to part "B", it is not clear why there are two equal parts, please explain.

Include the information published in the article: on the indole-3-acetamide-dependent (IAM) IAA production pathway in plants and properties of the proteins and genes involved “Thomas Lehmann, Maik Hoffmann, Mathias Hentrich, Stephan Pollmann, Indole-3-acetamide-dependent auxin biosynthesis: A widely distributed way of indole-3-acetic acid production? European Journal of Cell Biology, Volume 89, Issue 12, 2010, Pages 895-905, https://doi.org/10.1016/j.ejcb.2010.06.021.”

References

Update citation. https://doi.org/10.1016/j.ejcb.2010.06.021.

Author Response

Reviewer 2:

1) Write the scientific names (genus and species) in italics, for example: Arabidopsis thaliana, Medicago truncatula, etc.

Done as suggested.

2) In Figure 1. Paradigm shift in the IAOx-dependent auxin biosynthesis pathway: An evolving view. 1) The chemical formula for indole-3-acetonitrile (IAN) is incorrect; its molecular structure corresponds to that of indole-3-acetic acid (IAA). Correct the chemical structure.

We apologize for the oversight in the chemical structure annotations within our Figure. We have now carefully reviewed and verified all depicted chemical structures to ensure their accuracy.

3) Part "A" of Figure 1 is equal to part "B", it is not clear why there are two equal parts, please explain.

Thank you for bringing this point to our attention. Indeed, Panel A and Panel B are intended to represent the evolving paradigm of the IAOx-dependent IAA biosynthetic pathway. However, we acknowledge that the original Figure may not have clearly conveyed this distinction. We have now revised the Figures, with the updated pathway highlighted in red to enhance clarity and emphasis.

4) Include the information published in the article: on the indole-3-acetamide-dependent (IAM) IAA production pathway in plants and properties of the proteins and genes involved “Thomas Lehmann, Maik Hoffmann, Mathias Hentrich, Stephan Pollmann, Indole-3-acetamide-dependent auxin biosynthesis: A widely distributed way of indole-3-acetic acid production? European Journal of Cell Biology, Volume 89, Issue 12, 2010, Pages 895-905, https://doi.org/10.1016/j.ejcb.2010.06.021.”

References

Update citation. https://doi.org/10.1016/j.ejcb.2010.06.021.

We sincerely thank the reviewer for providing this reference. We have carefully reviewed the manuscript and have now integrated its relevant content into the revised text. The current description reads: “The traditional model is underpinned by multiple lines of evidence consolidating IAM and IAN as key intermediates [81]”

Ref:

Thomas Lehmann, Maik Hoffmann, Mathias Hentrich, Stephan Pollmann, Indole-3-acetamide-dependent auxin biosynthesis: A widely distributed way of indole-3-acetic acid production? European Journal of Cell Biology, 2010,89(12): 895-905

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is a high-quality, timely, and conceptually strong review of the IAOx-dependent auxin biosynthesis pathway. Its structure, scientific rigour, and clarity are commendable. The following minor suggestions are intended to further improve clarity, balance, and conceptual focus.

While the paradigm shift from a linear to a network-based IAOx model is convincingly argued, the authors are encouraged to more explicitly distinguish conclusions strongly supported by genetic and metabolomic evidence from those that remain hypothetical. In addition, it would be helpful to clarify that much of the mechanistic evidence derives from Brassicaceae species and that extrapolation to other taxa should be made with caution.
The absence of a clearly identified enzyme for direct IAOx-to-IAA conversion is a key issue. The discussion could be strengthened by briefly expanding on plausible biochemical mechanisms, including enzymatic, multi-enzyme, or compartment-dependent processes, and by clearly separating evidence-based models from speculation.
The proposed signalling roles of IAN and IAM are compelling. This section would benefit from a more focused discussion of tissue-specific functions, key experimental gaps, and concise suggestions for future experimental approaches to address these questions.
As metabolic channelling is central to the revised framework, the authors may consider more explicitly linking it to subcellular organisation and protein interactions and briefly discussing how developmental or stress contexts may dynamically reshape the IAOx network.
The authors may clarify how IAOx flux partitioning mechanistically underpins growth–defense trade-offs, and how the proposed network-based model revises traditional interpretations of hormone–defence crosstalk.

Overall, these minor revisions would further strengthen the manuscript’s clarity and conceptual impact, reinforcing its value as a reference work in auxin biosynthesis and metabolic regulation.

Author Response

Reviewer 3:

We sincerely thank you for your valuable time and insightful comments on our manuscript. Your feedback, particularly regarding the need to more explicitly distinguish strongly supported conclusions from hypothetical propositions and to clarify the species-specificity of the mechanistic evidence, has greatly helped us improve the clarity and precision of our review.

Below, we provide a point-by-point response to your constructive critique. In summary, we have thoroughly revised the manuscript by:

1) While the paradigm shift from a linear to a network-based IAOx model is convincingly argued, the authors are encouraged to more explicitly distinguish conclusions strongly supported by genetic and metabolomic evidence from those that remain hypothetical. In addition, it would be helpful to clarify that much of the mechanistic evidence derives from Brassicaceae species and that extrapolation to other taxa should be made with caution.

Thank you for your constructive feedback. We have carefully revised the manuscript to more clearly distinguish between well-supported conclusions and hypothetical proposals, and to clarify the species specificity of the mechanistic evidence. The main revisions are as follows:

Line 101-103: “It is important to note that the mechanistic insights and the paradigm shift discussed herein are primarily derived from studies in Brassicaceae species, notably Arabidopsis.”

Line 148-151: “These observations demonstrate that IAOx metabolism or perception can be activated in non-Brassicaceae species, yet the enzymatic basis for IAOx conversion to IAA in these species remains unclear and may not involve orthologs of the Arabidopsis CYP79B or the unknown enzyme implied by the sur2 studies.”

Line 201-205: “Through a comprehensive genetic approach involving the generation and analysis of higher-order mutants targeting entire gene families (cyp71a12 a13 a18, nit1 nit2 nit3 nit4, ami1 toc64-III toc64-V faah1 faah2), combined with detailed metabolomic profiling, Fenech et al. (2025) provided compelling evidence that challenges the established linear model.”

Line 270-272:“…inferred from the inability of mutation in CYP71A, NIT, and AMI families to suppress sur2 phenotypes…”

Line 277-279: “Crucially, the discussions below on mechanisms and origins are informed speculations derived from established evidence, not extensions of the evidence itself.”

Line 319: “The IAOx pathway, as currently understood from genetic dissection in Arabidopsis,…”

These revisions ensure that the review more accurately reflects the strength of the evidence, clearly distinguishes established findings from forward-looking hypotheses, and responsibly communicates the scope and limitations of current mechanistic understanding, particularly regarding its foundation in Brassicaceae biology.

Thank you again for your valuable suggestions, which have significantly improved the clarity and rigor of our manuscript.

2) The absence of a clearly identified enzyme for direct IAOx-to-IAA conversion is a key issue. The discussion could be strengthened by briefly expanding on plausible biochemical mechanisms, including enzymatic, multi-enzyme, or compartment-dependent processes, and by clearly separating evidence-based models from speculation.

We sincerely thank the reviewer for this insightful comment, which directly addresses the core challenge and future direction highlighted by the recent paradigm shift. We fully agree. In our revised manuscript, we have taken two concrete steps to address this point, both deeply informed by the definitive evidence presented in Fenech et al. (2025):

(1) Clear demarcation between evidence-based conclusions and informed speculation

we have fundamentally restructured the narrative flow at the transition between the results summary (Section 3) and the future perspectives (Section 4), which is described as follows:

Line 244-263: “In summary, the work by Fenech et al. presents a compelling rebuttal of the linear IAOx pathway model and establishes a robust, evidence-based framework for understanding IAOx metabolism in Arabidopsis[36]. This framework is founded on three key pillars supported by integrated genetic, pharmacological and metabolomic data: 1) the existence of an efficient alternative pathway from IAOx to IAA that is hyperactivated in the sur2 mutant; 2) the operational independence of this pathway from the CYP71A, NIT, AMI/TOC64/FAAH and IAMH gene families; and 3) the parallel and metabolically decoupled roles of IAOx, IAN and IAM as precursors, each engaging tissue-specific routes to produce IAA via distinct primary mechanisms—an unknown enzyme for IAOx, NITs for IAN and IAMHs for IAM. [30,35,40,41]. This evidence-based model conclusively defines what is known—the existence and independence of this pathway, while starkly revealing what remains unknown: the biochemical identity of the conversion mechanism itself.”

Line 277-279: “Crucially, the discussions below on mechanisms and origins are informed speculations derived from established evidence, not extensions of the evidence itself.”

(2) Expansion on plausible biochemical mechanisms for the unknown conversion step, which is described as follows: “While the genetic and metabolomic evidence unequivocally demands the existence of an efficient IAOx-to-IAA conversion route, its biochemical mechanism remains unresolved. Several plausible hypotheses merit consideration: a single unidentified enzyme (e.g., a novel oxidoreductase, lyase or dioxygenase) could catalyze direct conversion; a multi‑enzyme metabolon—akin to the camalexin biosynthetic complex—might channel IAOx through transient association of catalytic and auxiliary subunits; strict subcellular compartmentalization (e.g., in peroxisomes or specialized ER domains) could provide both the requisite catalytic milieu and regulatory isolation; or an enzyme‑triggered chemical step might generate a reactive intermediate that rapidly rearranges to IAA in the cellular context. These speculative models are grounded in the logical imperative of the genetic data and in known biochemical principles, and their experimental dissection represents the immediate biochemical frontier in the field.”

Thank you again for this valuable suggestion.

3) The proposed signalling roles of IAN and IAM are compelling. This section would benefit from a more focused discussion of tissue-specific functions, key experimental gaps, and concise suggestions for future experimental approaches to address these questions.

Thank you for your careful review of our manuscript and for your valuable feedback regarding the section on the “signaling roles of IAN and IAM.” We agree with your assessment that this part would benefit from a more focused discussion on tissue-specific functions, identification of key experimental gaps, and clear suggestions for future experimental approaches.

We have revised the manuscript accordingly, incorporating the following additions and modifications to address your suggestions, which is described as follows:

“i) The central enigma: The unidentified enzyme(s) catalyzing IAOx-to-IAA conversion and its regulation

The most pressing unresolved question, revealed by the work of Fenech et al. (2025), is the molecular identity of the…phylogenetic-functional analyses across monocot and eudicot lineages to trace the evolution of IAN/IAM-mediated signaling modules.”

4) As metabolic channelling is central to the revised framework, the authors may consider more explicitly linking it to subcellular organisation and protein interactions and briefly discussing how developmental or stress contexts may dynamically reshape the IAOx network.

We sincerely thank the reviewer for this valuable suggestion. In line with your recommendation, we have supplemented the section titled “iii) The regulatory logic of metabolic channeling and the determinative role of cell identity” with the following specific additions: “Critically, metabolic channeling is not a static configuration but is dynamically reshaped by developmental and environmental cues. For example, under pathogen attack, transcriptional reprogramming mediated by WRKY33 may suppress the unknown IAOx-to-IAA converting enzyme while upregulating CYP71A12/13, thereby redirecting IAOx flux toward camalexin synthesis [54]. Such dynamic repartitioning of metabolic flux likely involves rapid post-translational modifications, allosteric regulation, or the reversible assembly of enzyme complexes. Furthermore, this channeling is likely underpinned by precise subcellular organization and protein–protein interactions. The biosynthesis of camalexin, for instance, involves a metabolon comprising CYP71A12, CYP71A13, GGP1, and GSTU4, which spatially coordinates intermediate transfer and prevents diffusion of reactive metabolites [68]. By analogy, the unknown route from IAOx to IAA may operate within dedicated subcellular niches—such as specialized endoplasmic reticulum domains or peroxisomes—or through transient enzyme complexes that physically segregate auxin production from defense-related pathways. Future research must leverage cutting-edge technologies such as single-cell metabolomics and spatial transcriptomics to construct a high-resolution spatiotemporal atlas of the IAOx metabolic network in planta. Integrating these approaches will be essential to map context-dependent channeling events and elucidate how plants toggle between metabolic programs in real time.”

5) The authors may clarify how IAOx flux partitioning mechanistically underpins growth–defense trade-offs, and how the proposed network-based model revises traditional interpretations of hormone–defence crosstalk.

Overall, these minor revisions would further strengthen the manuscript’s clarity and conceptual impact, reinforcing its value as a reference work in auxin biosynthesis and metabolic regulation.

We sincerely thank the reviewer for raising this pivotal question. We fully agree that elucidating the mechanistic basis of IAOx flux partitioning and its implications for revising the theoretical framework of hormone–defense crosstalk constitutes a critical component of this work. Accordingly, we have substantially expanded the section “v) Network integration and signal crosstalk under environmental stress” to systematically address this point. The key additions and revisions include the following: “In this context, the allocation of IAOx flux provides a mechanistic basis for classical growth–defense trade-offs. Rather than viewing IAA and defense metabolites as endpoints of a single linear pathway,…Thus, crosstalk is not an obligatory metabolic linkage but a competitive, regulatory relationship centered on the IAOx branch point. Hormone and defense outputs can be coordinated or antagonized through transcriptional and allosteric control of the enzymes that compete for IAOx, enabling a more flexible and context-dependent balance between growth and defense.”

We believe that through the aforementioned additions and revisions, the manuscript now provides a comprehensive and substantive response to the concerns raised. We sincerely hope that these modifications meet with your approval.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Journal Plants (ISSN 2223-7747)

Manuscript ID plants-4013468

Type Review

Title The IAOx-Dependent IAA Biosynthesis Pathway: Established Understanding, Paradigm Shifts, and Future Challenges

Authors Ming-Kun Ma , Verena Kriechbaumer * , Dong-Wei Di *

Section Plant Molecular Biology

Special Issue Advances in Plant Auxin Biology

Dear Editor

In its current form, the manuscript can proceed to further stages of editing. I would like to mention for future reference that it is difficult to check the text (compare the first version with the second) without marking the changes in red.

Congratulations to the authors.

Best regards

Reviewer

Please pay attention to the editing of the text, as the thematic paragraphs have become confused.
There are still errors in the subsection - REFERENCES.
The Latin names of plant genera and species should be written in italics, etc.