Auxin Biosynthesis, Transport, Signaling, and Its Roles in Plant Leaf Morphogenesis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

-1. The manuscript reads more like a compact auxin textbook plus a relatively brief, Arabidopsis-centric summary of leaf morphogenesis. 
1) The central integrative thread, how specific features of auxin homeostasis and signaling mechanistically map onto concrete leaf traits, is not always explicit.
2) The first ~2-3 paragraphs devote substantial space to general auxin history and broad physiological roles, but relatively little to the specific conceptual problems in leaf morphogenesis that remain unresolved. 
3) What is the specific gap compared with previous reviews? What do you think?

- 2. The Introduction states that this paper "provides a comprehensive review of recent research on auxin synthesis, transport, and signal transduction" and then applies this to leaf morphogenesis. 
1) Given that several reviews already exist on auxin and leaf development, could the authors explicitly explain what is new here (e.g., inclusion of recently discovered ABL / GLP-TMK modules, or explicit stage-wise mapping of auxin functions during leaf development)?

2) "Auxin constitutes a class of endogenous hormones characterized by the presence of an unsaturated aromatic ring and an acetic acid side chain, with indole-3-acetic acid being its chemical nature". This is confusing: auxin is a class of molecules, not "being IAA in its chemical nature", and PAA/4-Cl-IAA already violates the strict "indole" description. Consider rephrasing this to avoid implying that all auxins are structurally identical to IAA.

- 3. Many subsections (especially biosynthesis and signaling) appear strongly Arabidopsis-centric, whereas some leaf morphogenesis examples extend to other species. 
1) Was this deliberate, or simply a reflection of where the literature is densest?
2) A short paragraph clarifying these points would make the review more transparent and help readers understand potential biases in species, gene families, and processes covered.

4. Table 1 and much of Section 3 emphasize auxin biosynthesis mutants with diverse developmental phenotypes (root traits, floral organs, stress responses, etc.), but many are only loosely tied to leaf developmental outcomes. 
1) Would it be possible to prioritize or highlight mutants with clear leaf phenotypes (e.g., rosette curvature, lamina size, leaf number, polarity defects), and more briefly summarize others?
2) Alternatively, could you add a concise sub-summary at the end of Section 3 explicitly explaining which biosynthetic nodes are most relevant for leaf development (for example, why spatiotemporal YUC expression is particularly important for leaf lamina growth)?

- 5. Could the authors more clearly distinguish established routes (e.g., IPyA pathway) from hypothesized or controversial steps (e.g., TAM-NHT, TRP-independent routes), so that readers do not overinterpret uncertain pathways as settled facts?
1) Is there a way to connect these uncertainties to leaf morphogenesis (e.g., do we have any evidence that non-canonical pathways contribute specifically to local auxin peaks in emerging leaf primordia)?

- 6. You mention that the IAOx route is Brassicaceae-specific yet emphasize its importance in plant defense and IAA production. 
1) It would help to explicitly clarify to what extent these conclusions can be generalized beyond Arabidopsis.

-7. Much of the transport/signaling discussion focuses on roots (gravitropism, lateral roots) or general developmental phenotypes without clearly linking them back to leaf morphogenesis. 
1) For instance, PIN2 / root agravitropism, AUX1 / LAX root phenotypes, and AFB1's role in root rapid responses occupy substantial space. 

-8. The manuscript describes ABP1's uncertain role and the discovery of ABL1 / 2 as auxin-binding GLP proteins that interact with TMKs, suggesting they may be the primary apoplastic auxin receptors. 
1) Given the longstanding controversy around ABP1, readers would benefit from a brief, explicit statement on the current consensus. 
2) "Genetic loss-of-function abp1 mutants show no dramatic phenotypes, so ABP1 is now considered dispensable in many contexts; ABL1 / 2 have emerged as stronger candidates for apoplastic auxin perception.…" This would avoid confusion for non-specialists.

- 9. Sections 6.1-6.3 are organized around stages (initiation, polarity, size/shape), but within each stage, the text sometimes reads as a gene list rather than a mechanistic synthesis. 
1) Could the authors more explicitly state, at the end of each subsection, what the prevailing mechanistic model is? For example, "Local auxin maxima, established by PIN1 and stabilized by AUX1 / LAX, define primordium positions and simultaneously repress KNOX1, thus committing cells to leaf fate".

- 10. Almost all mechanistic details are from Arabidopsis. While understandable, the journal’s readership includes many crop scientists.
1) Are there examples where auxin manipulations in crops (e.g., CRISPR editing of ARFs or PINs) have led to altered leaf architecture that improves yield or stress tolerance?

- 11. Auxin does not act in isolation during leaf development, yet the section on leaf morphogenesis is almost exclusively auxin-centric.
1) Would it strengthen the review to briefly discuss interaction with cytokinin, gibberellin, or brassinosteroids in shaping leaf size and complexity? 
2) Even a short paragraph referencing key cross-talk nodes would help situate auxin within the broader hormonal network.
3) Similarly, how do environmental signals (light, mechanical forces, nutrient status) feed into the auxin networks described here to modulate leaf morphology?
4) Could the authors more clearly explain why this network is an instructive example of auxin-gene interactions in morphogenesis (e.g., demonstrating how auxin maxima and CUC repression define repeated marginal outgrowths)?
5) Are there any known links between these margin networks and leaf function (e.g., boundary layers, light interception, hydraulic conductance), or is this mostly a patterning problem at present?

- 12. Could the authors briefly comment on recent modeling or live-imaging approaches that have advanced understanding of auxin-driven leaf morphogenesis, and how future work might combine genetics, imaging, and modeling?

-13. Since the Introduction emphasizes the agronomic importance of leaf architecture in cereals and leafy vegetables, it would be valuable if the explicitly addresses how these auxin networks might be exploited in breeding or engineering. For instance, are there plausible targets (e.g., moderate modulation of ARF2 / 3 / 4, or PIN1 expression dynamics) that could tune leaf angle or lamina size without severe pleiotropy?
1) If so, what are the main risks and trade-offs of manipulating auxin pathways for crop improvement?
2) The general facts about auxin (biosynthesis pathways, transporters, central role in growth and differentiation) and notes, in broad terms, that gaps remain regarding specific genes and interactions. 

- 14. It also mentions that the mechanisms defining auxin convergence points at the apex and margin remain an elusive, genuinely important open question. 
1) Also, many sentences could appear in any auxin review ("Auxin is the most abundant and crucial hormone in plants", etc.) and do not highlight what is specific to leaf morphogenesis in this manuscript. 

- 15. The text mentions "unresolved gaps" but does not clearly prioritize them or suggest approaches. 

-16. The conclusions state that leaves are essential for photosynthesis, but do not really connect specific auxin-dependent morphological traits to performance (e.g., light interception, gas exchange, hydraulic conductance). 

17. A short paragraph linking the morphogenetic perspective to functional consequences (and thus crop improvement) would provide a more compelling closure.

-18. It might also be helpful to complement the largely Arabidopsis-focused sections with one or two applied horticultural examples where auxin is already being manipulated in practice. For instance, recent studies on auxin-driven adventitious rooting and morphophysiological responses in some ornamental crops (e.g., https://doi.org/10.7235/HORT.20250032) could be briefly linked as case studies. Linking the mechanistic auxin framework in this review to such examples would help illustrate its relevance across different crop types and gently broaden the applied scope of the manuscript.

- 19. Keywords need to be rearranged alphabetically.

Thank you.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

plants-4040715

Auxin biosynthesis, transportation, signaling, and its roles in plant leaf morphogenesis

 

Auxin is an important class of phytohormone participating in various stages of plant growth and development. Auxins play a cardinal role in the coordination of many growth and behavioral processes in plant life cycles and are essential for plant body development. Moreover, the (dynamic and environment-responsive) pattern of auxin distribution within the plant is a key factor for plant growth, its reaction to its environment, and specifically for the development of plant organs.

 

While it plays crucial roles in the biosynthesis, signal transduction processes, and transport, including an important role in multiple processes governing leaf morphogenesis, a topic highlighted and discussed in the following article.

Through this extensive literature review, the authors provide insights into the recent progress and research on auxin biosynthesis, metabolism, transport mechanisms, and associated signalling pathways.

 

For the improvement of the article, some suggestions and queries are discussed:

 

Considering the extensive scope and function of the plant hormone, studies have extensively investigated its biosynthesis and role in plant growth and development. Key literature has discussed the mechanisms of auxins in plant development (Vanneste et al, 2025; Nature Reviews Molecular Cell Biology; Hou et al, 2025 Frontiers in Plant Science; Gao et al, 2024 Plants, including others).

How does the present literature contribute to improving our knowledge in this area? What is the major highlights and novel concepts discussed?

Another literature analysis on this key phytohormone is significant only if it provides novel insights and perspectives on emerging advances in less explored themes connected with auxins. Instead of focusing on multiple areas- auxin types, their functions, biosynthetic pathways, transport, signaling and others (since this is already known), it is suggested to highlight and discuss a new theme or emerging concepts.

 

Abstract, Line 23-26: Additionally, we summarize the significant contributions…………….in plant development. The abstract of a manuscript provides a summarized account of a topic, discussing its significance, insights, emerging trends, advances, and challenges, if any. In the abstract, authors should discuss key examples of recent advances, future directions, and prioritize actionable goals. Nowhere has this been discussed.

 

Line 46-48: Based on these discoveries, ……………….20^th century. In the introduction, the chemical properties and biological activities of the auxin hormone should be discussed briefly, giving examples.

 

In most sections of the article, different aspects of the role of auxins is discussed, providing specific examples of research studies. However, the article seems to be a mere compilation of the existing literature, providing no new concepts or the author's perspective in this direction. An interesting literature theme provides novel hypotheses and conclusions by highlighting progress made and the way forward.

 

 

 

 

 

 

 

Comments on the Quality of English Language

The English language could be improved for better expression of research and clarity.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript by Zheng et al. provides a systematic review of auxin biosynthesis, transport, and signaling, and its role in leaf morphogenesis. While the summary of auxin's function in leaf development is a notable strength, the coverage of recent advances in auxin biosynthesis and signaling is insufficient. Specific points are detailed below:

1) Lines 55–57: While it is widely accepted that IBA functions by conversion to IAA via β-oxidation, the question of whether IBA acts as an auxin per se or solely as an IAA precursor remains open. The authors should maintain a more cautious stance on this point.

2) Lines 64–67 and 166–169: Although less studied, key genes in the Trp-independent pathway, such as INS, have been identified. It is established that indole serves as the branch point between the Trp-dependent and -independent pathways. Therefore, the dashed box in Figure 2 seems unwarranted. Furthermore, the intermediate steps from chorismate to indole have been reviewed systematically. Relevant literature should be consulted:

Wang B et al. Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis. Proc Natl Acad Sci USA 2015, 112(15):4821–4826.

Di, D. W. et al. The biosynthesis of auxin: how many paths truly lead to IAA?. Plant Growth Regulation 2016, 78(3): 275-285.

3) Lines 84–85: The literature on TMK–ABL3 regulating PIN2 should be cited and discussed.

Rodriguez, L. et al. ABP1/ABL3-TMK1 Cell-Surface Auxin Signaling Targets PIN2-Mediated Auxin Fluxes for Root Gravitropism. Cell 2025, 188, 6138-6150.e17.

4) Line 162: The full names of ASA and ASB should be provided at their first mention.

5) Table 1, The authors have compiled a useful summary of recently reported key genes involved in auxin biosynthesis. However, notable omissions exist:

YUC8 is currently the only reported Arabidopsis YUC mutant that exhibits a pronounced phenotype even as a single mutant. The relevant reference should be included:

Di, D. W et al. Functional roles of Arabidopsis CKRC2/YUCCA8 gene and the involvement of PIF4 in the regulation of auxin biosynthesis by cytokinin. Scientific Reports 2016, 6, 36866.

Recent findings have brought a paradigm shift in the IAOx pathway, suggesting that previously considered key downstream genes, such as AMI1 and NITs, may not play essential roles. This should be addressed in the text with citation:

Fenech, M. et al. The CYP71A, NIT, AMI, and IAMH Gene Families Are Dispensable for Indole-3-Acetaldoxime-Mediated Auxin Biosynthesis in Arabidopsis. Plant Cell 2025, 37, koaf242.

6) Lines 309–330: The canonical TIR1/AFB-Aux/IAA signaling pathway has recently undergone a paradigm shift with the discovery of cAMP as a pivotal second messenger. This section requires substantial reorganization and discussion, and Figure 3 should be updated accordingly. The following key references must be incorporated:

Qi, L. et al. Adenylate Cyclase Activity of TIR1/AFB Auxin Receptors in Plants. Nature 2022, 611, 133-138.

Chen, H. et al. TIR1-Produced cAMP as a Second Messenger in Transcriptional Auxin Signalling. Nature 2025, 640, 1011-1016.

7) Lines 357–358: Recent research highlights the significant role of ABL3. The authors should cite and discuss this:

Rodriguez, L. et al. ABP1/ABL3-TMK1 Cell-Surface Auxin Signaling Targets PIN2-Mediated Auxin Fluxes for Root Gravitropism. Cell 2025, 188, 6138-6150.e17.

8) While the role of auxin in leaf morphogenesis is well summarized, the manuscript lacks a schematic Figure. It is recommended that the authors include a summary diagram illustrating the regulatory network of auxin in leaf development.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

-1. "Transportation" in the title is unusual; "transport" is standard in auxin literature. Given the very technical level of the review, this word choice stands out and feels slightly non-idiomatic.

2. The Abstract claims that the elucidated network already "serves as a blueprint for rationally designing crop architecture" and that translating this into precision breeding pipelines is a "direct pathway" to engineering next-generation crops.
1) Yet later sections give almost no concrete examples of successful crop breeding programs that have used these auxin nodes to modify leaf traits.

- 3. Many phenotypes in Table 1 are root-related, stress-related, or general growth traits. Only a subset clearly relates to leaf form (e.g., YUCCA mutants with leaf and margin defects).
1) Could Table 1 be reorganized or annotated to highlight which genes are directly implicated in leaf traits (leaf size, serration, polarity), and which are more general?

- 4. You correctly describe the classical double-negative Aux / IAA-ARF model and then state that TIR1 AC activity and local cAMP production are required for transcriptional responses in vivo, even when Aux/IAA degradation still occurs.
1) Mechanistically, do you propose that cAMP is now considered a co-signal on par with Aux / IAA degradation, or that it acts as an obligatory cofactor modifying ARF activity?
2) How do you envision this new branch changing our interpretation of earlier genetic data on leaf phenotypes in arf / aux-iaa mutants? Some explicit commentary here would be valuable.

- 5. The section 6 opening paragraphs discuss coordinated roles of auxin with CK, GA, BR, SL, ABA, JA, and SA under both normal and stress conditions, in quite broad terms.
1) This reads more like a general "auxin and other phytohormones in plant stress and development" overview than a focused discussion on leaf morphogenesis.
2) Could you sharpen this part by explicitly choosing leaf-specific examples of hormone crosstalk (e.g., leaf size under drought, shade-induced leaf morphology), and more tightly linking each hormonal interaction back to the three stages of leaf development you defined?

-6. You explicitly state that the review intentionally focuses on Arabidopsis.
1) Yet you then speak broadly about "designing crop architecture" amd "next-generation crops" as if the modules were already validated and transferable. What do you think?

- 7. The manuscript is acronym-dense (TAM, IAM, IAOx, IPyA, NHT, etc.). A summary table of abbreviations specific to auxin pathways (in addition to the general list at the end, if any) could help readability, especially for readers coming from more applied fields.

Thank you.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript may be considered in the present form.

Author Response

We would like to express our sincere gratitude to the reviewer for their time and expertise in evaluating our manuscript, as well as for their positive feedback recommending that the manuscript be considered for publication in its current form. We greatly appreciate their comprehensive review and constructive suggestions throughout the revision process.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have answered all my concerns.

Author Response

We would like to express our sincere gratitude to the reviewer for their comprehensive and constructive evaluation of our manuscript, as well as for confirming that we have effectively addressed all of their concerns. We greatly appreciate the time and expertise they have dedicated to enhancing the quality of our work.