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  • Tingting Xu,
  • Zengwei Yang and
  • Qingmin Xie *
  • et al.

Reviewer 1: Anonymous Reviewer 2: Olga Fedotova Reviewer 3: Irina Dodueva

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The terminology should be revised to distinguish between the hormonal pathways identified through transcriptomic analysis and the compounds applied experimentally. Auxin and jasmonic acid are plant hormones, whereas NPA is a synthetic inhibitor of polar auxin transport. MeJA is a bioactive methylated derivative of jasmonic acid and was the compound applied exogenously. Therefore, “jasmonic acid,” “methyl jasmonate,” and “jasmonate signaling” should not be used interchangeably. The title and conclusions should explicitly refer to MeJA when describing the experimental treatment and to jasmonate signaling when discussing the associated molecular pathway.

suggestion:

Roles of an auxin transport inhibitor and methyl jasmonate in waterlogging-induced aerenchyma formation in Luffa.

The conclusions are generally consistent with the abstract regarding aerenchyma formation in ‘Zhonglü’, the involvement of auxin- and jasmonate-related signaling, and the effects of NPA and MeJA. However, some inconsistencies should be addressed. The contrasting response of ‘Yalü’, which represents a central finding in the abstract, should also be included in the conclusions. Conversely, the statement that Luffa has several entry points for oxygen is not clearly supported by the findings summarized in the abstract and should either be substantiated with specific evidence or removed. In addition, the proposed convergence of auxin transport and MeJA signaling through ARF18, IAA32, GH3, and TIFY9 should be presented cautiously unless direct functional evidence is available. These genes would be more appropriately described as candidate genes associated with hormonal regulation of aerenchyma formation.

Author Response

Responses to Reviewers’Comments

Referee: 1

The terminology should be revised to distinguish between the hormonal pathways identified through transcriptomic analysis and the compounds applied experimentally. Auxin and jasmonic acid are plant hormones, whereas NPA is a synthetic inhibitor of polar auxin transport. MeJA is a bioactive methylated derivative of jasmonic acid and was the compound applied exogenously. Therefore, “jasmonic acid,” “methyl jasmonate,” and “jasmonate signaling” should not be used interchangeably. The title and conclusions should explicitly refer to MeJA when describing the experimental treatment and to jasmonate signaling when discussing the associated molecular pathway.

suggestion:

Roles of an auxin transport inhibitor and methyl jasmonate in waterlogging-induced aerenchyma formation in Luffa.

The conclusions are generally consistent with the abstract regarding aerenchyma formation in ‘Zhonglü’, the involvement of auxin- and jasmonate-related signaling, and the effects of NPA and MeJA. However, some inconsistencies should be addressed. The contrasting response of ‘Yalü’, which represents a central finding in the abstract, should also be included in the conclusions. Conversely, the statement that Luffa has several entry points for oxygen is not clearly supported by the findings summarized in the abstract and should either be substantiated with specific evidence or removed. In addition, the proposed convergence of auxin transport and MeJA signaling through ARF18, IAA32, GH3, and TIFY9 should be presented cautiously unless direct functional evidence is available. These genes would be more appropriately described as candidate genes associated with hormonal regulation of aerenchyma formation.

Responds: We sincerely thank the reviewer for highlighting the important distinction between endogenous hormonal pathways and exogenously applied compounds, as well as for the thoughtful suggestions regarding data interpretation. We have carefully revised the manuscript accordingly:

  1. Clarification of terminology (MeJA vs. JA / MeJA vs. jasmonate signaling)

As suggested, we now strictly distinguish between methyl jasmonate (MeJA) as the experimentally applied compound and jasmonate signaling when referring to the inferred molecular pathway. All instances where “jasmonic acid”, “methyl jasmonate”, and “jasmonate signaling” were previously used interchangeably have been corrected throughout the manuscript, including the Title, Abstract, Results, and Discussion.

Specifically:

(1) The title has been revised to “Roles of an auxin transport inhibitor and methyl jasmonate in waterlogging‑induced aerenchyma formation in Luffa”

(2) Experimental treatments are now consistently referred to as NPA and MeJA.

(3) Transcriptome-based inferences are described using terms such as auxin-related pathways, jasmonate signaling, or jasmonate-responsive regulation.

  1. Revision of the Conclusions section

The Conclusions have been fully revised: the contrasting ‘Yalü’ response is now included; remove the unsupported statement regarding “multiple oxygen entry points”, since this inference is not directly demonstrated by the current dataset; and ARF18, IAA32, GH3, and TIFY9 are described as candidate genes within a broader waterlogging-induced syndrome (aerenchyma + adventitious roots), consistent with the absence of direct functional evidence.

We have rewritten the Conclusions as follows:

In summary, waterlogging induced contrasting responses in two luffa cultivars: ‘Zhonglü’ formed aerenchyma in the hypocotyl cortex together with profuse adventitious roots, whereas ‘Yalü’ was not. Transcriptomic profiling highlighted auxin- and jasmonate-associated DEGs, including ARF18, IAA32, GH3, and TIFY9, which are proposed here as candidate genes potentially linking hormonal pathways in aerenchyma development. Exogenous application of 5 μmol/L NPA and 20 μmol/L MeJA modestly enhanced aerenchyma formation in ‘Zhonglü’ under flooded conditions, suggesting that polar auxin transport and jasmonate-related signaling modulate this trait. Anatomical evidence indicates aerenchyma may contribute to internal oxygen diffusion within the hypocotyl, though the full pathway network requires tissue-specific validation. However, the detailed processes involved in the formation of epidermal slits in the hypocotyl and adventitious root were not investigated in this study, so further research is needed.

  1. Caution in mechanistic claims

In the absence of direct genetic or biochemical validation, we no longer imply functional convergence but instead state that these DEGs represent candidate genes associated with hormonal regulation of aerenchyma development, meriting further functional characterization.

We believe these revisions have improved the precision of our terminology and the rigor of our conclusions, and we greatly appreciate the reviewer’s guidance in strengthening the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript ID

horticulturae-4428806

Title

Roles Of Auxin Transport Inhibitor And Jasmonic Acid In Waterlogging-induced Aerenchyma Formation In Luffa

 

Waterlogging limits plant growth and productivity. Therefore, studying the mechanisms that contribute to increasing resistance to waterlogging in plants is undoubtedly relevant. Aerenchyma formation, regulated internodal elongation, and random rooting help plants grow in soils with low oxygen levels. The authors have done a good job and elucidate the key genes involved in aerenchyma development and advance understanding of the molecular mechanisms underlying hormonal regulation in Luffa.

This manuscript undoubtedly has scientific value. It is well written and gets to the heart of the research problem being explored. However, the authors should consider a number of recommendations and answer questions presented below.

  1. According to the journal guidelines, Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. Please make corrections to the text of the figure notes.

Abstract

  1. Line 17. "(DEGs)". I recommend removing the abbreviation, as it is not used further in the abstract.
  2. Lines 19-20. I recommend editing the sentence. The abbreviation "NPA" needs to be expanded. "N-1-naphthylphthalamic acid".
  3. Line 20. "MeJA". I recommend adding a definition of the abbreviation.

Introduction

  1. Lines 52-61. You noted that aerenchyma can be divided into inducible and constitutive, and indicated which plants possess which of these types. I would also recommend mentioning the object of your research—luffa.
  2. Lines 72, 79, 204, 207 and etc. I recommend not highlighting definition of abbreviations in a different font.
  3. Line 80. I recommend adding information about where the luffa is used and explaining why learning about the luffa is important.
  4. Lines 83-92. I recommend editing and shortening the description of the research results in the introduction.

Materials and Methods

  1. Line 96. I recommend adding information about the research object here: Luffa (Luffa cylindrica)
  2. Line 97. Why were these particular Luffa cultivars chosen?
  3. Line 102. I recommend indicating the manufacturer of the growth chamber.
  4. Line 103. Please check the spelling of the degree Celsius symbol.
  5. Line 107. I recommend that you describe in more detail how you determined root activity and, if possible, provide a link to the reference. Please include the definition of TTC method.
  6. Line 111. "MTH-1". I recommend adding a definition of the abbreviation.
  7. Line 113. I recommend indicating the manufacturer of the optical microscope.
  8. Line 124. Why was transcriptional analysis only done on Zhonglü luffa cultivar?
  9. Lines 129, 185, 224, 311. I recommend writing the p-value in lowercase letters.
  10. Line 142. I recommend indicating how your data are presented (means ±SD), how many independent experiments were conducted, and what p-value indicates significant differences (p<0.05). In the figure notes (Lines 183, 222, 309), I recommend indicating the statistical analysis method or test you used.

 Results

  1. Line 146. I recommend replacing “Morphological Changes” with “Morphological changes”.
  2. Line 159. ‘YL’ and ‘ZL’. I recommend adding definitions for these abbreviations. According to the journal rules, abbreviations should be expanded upon their first mention in a figure.
  3. Line 183. CK-ZL, WL-ZL, CK-YL, WL-YL. I recommend adding definitions for these abbreviations. In the figure note, I recommend indicating the statistical analysis method or test you used.
  4. Lines 202-205. I recommend editing or moving the discussion of the data from the results subchapter to the discussion chapter.
  5. Line 225. I recommend replacing “Effects of NPA and MeJA Application on aerenchyma formation Under waterlogging Stress” with “Effects of NPA and MeJA application on aerenchyma formation under waterlogging stress”.
  6. Line 294. Unfortunately, I could not find the definition of the TIFY9 gene in the manuscript. What is this gene, and what protein does it encode?
  7. Line 309. I recommend adding an explanation of the abbreviations for the genes being studied in the figure's note.

 

 

Author Response

Responses to Reviewers’Comments

Referee: 2

Manuscript ID

horticulturae-4428806

Title

Roles Of Auxin Transport Inhibitor And Jasmonic Acid In Waterlogging-induced Aerenchyma Formation In Luffa

Waterlogging limits plant growth and productivity. Therefore, studying the mechanisms that contribute to increasing resistance to waterlogging in plants is undoubtedly relevant. Aerenchyma formation, regulated internodal elongation, and random rooting help plants grow in soils with low oxygen levels. The authors have done a good job and elucidate the key genes involved in aerenchyma development and advance understanding of the molecular mechanisms underlying hormonal regulation in Luffa.

This manuscript undoubtedly has scientific value. It is well written and gets to the heart of the research problem being explored. However, the authors should consider a number of recommendations and answer questions presented below.

  1. According to the journal guidelines, Acronyms/Abbreviations/Initialisms should be defined the first time they appear in each of three sections: the abstract; the main text; the first figure or table. Please make corrections to the text of the figure notes.

Responds: We thank the reviewer for reminding us of the journal’s abbreviation policy. We have carefully revised the manuscript to ensure that all acronyms/abbreviations are defined at first mention in each of the three required sections:

Abstract: full expansions provided for NPA, MeJA, and DEGs upon first use;

Main text: each abbreviation is spelled out again at its first appearance in the Introduction/Results;

First figure legend definitions have been added to the first figure note.

  1. Line 17. "(DEGs)". I recommend removing the abbreviation, as it is not used further in the abstract.

Responds: Thank you for the suggestion. We have removed the abbreviation “(DEGs)” from Line 17 of the Abstract.

  1. Lines 19-20. I recommend editing the sentence. The abbreviation "NPA" needs to be expanded. "N-1-naphthylphthalamic acid".

Responds: Thank you for the suggestion. The full name N-1-naphthylphthalamic acid (NPA) has been provided at its first appearance in the Abstract.

  1. Line 20. "MeJA". I recommend adding a definition of the abbreviation.

Responds: Thank you for the suggestion. The abbreviation has been expanded to methyl jasmonate (MeJA) in the Abstract.

Introduction

  1. Lines 52-61. You noted that aerenchyma can be divided into inducible and constitutive, and indicated which plants possess which of these types. I would also recommend mentioning the object of your research—luffa.

Responds: Thank you for the suggestion. We have added a brief sentence noting that Luffa exhibits inducible aerenchyma in roots, linking the general classification to our study species. It has been added as follows:

In herbaceous dicots, including Luffa cylindrica, aerenchyma typically initiates in the outer cortex of the hypocotyl (the non-woody “bark” region beneath the epidermis), rather than from vascular or pith tissues, representing an inducible type that mitigates oxygen deficit.

  1. Lines 72, 79, 204, 207 and etc. I recommend not highlighting definition of abbreviations in a different font.

Responds: Thank you for the suggestion. All abbreviations/acronyms have been unified to the standard font.

  1. Line 80. I recommend adding information about where the luffa is used and explaining why learning about the luffa is important.

Responds: Thank you for the suggestion. A short statement on the economic and ecological importance of Luffa cylindrica has been added as follows:

Luffa (Luffa cylindrica L.), a widely cultivated Cucurbitaceous vegetable in subtropical and tropical Asia, is valued both as an edible crop and for its dried fibrous vascular skeleton, which is used industrially as natural scrubbers and biomaterials.

 

  1. Lines 83-92. I recommend editing and shortening the description of the research results in the introduction.

Responds: Thank you for the suggestion. The description of the research results in the introduction has been revised as follows:

In particular, whether polar auxin transport and jasmonate signaling coordinate aerenchyma formation in the hypocotyl is unclear. The present study therefore compared two contrasting cultivars and integrated anatomical observation, hormone treatments, and transcriptomic analysis to identify candidate genes and hormonal pathways potentially involved in luffa’s flooding adaptation.

Materials and Methods

  1. Line 96. I recommend adding information about the research object here: Luffa (Luffa cylindrica)

Responds: Thank you for the suggestion. We agree and have revised the beginning of the Materials and Methods section as follows:

Luffa (Luffa cylindrica L.) seedlings were used in this study.

  1. Line 97. Why were these particular Luffa cultivars chosen?

Responds: We thank the reviewer for this question. The two cultivars were not selected arbitrarily. ‘Zhonglü’ (a Shouguang-type commercial cultivar) is described by seed catalogs and local cultivation guides as heat-tolerant and waterlogging-tolerant, whereas ‘Yalü’ (a Yalü cultivar from Guangdong Kenong) is reported to have moderate waterlogging tolerance. In a preliminary 14-d pot screening, ‘Zhonglü’ consistently produced hypocotyl aerenchyma and profuse adventitious roots under flooding, while ‘Yalü’was not. We therefore chose this pair to maximize phenotypic contrast and to allow transcriptomic capture of the induced aerenchyma program in the responsive genotype, while using the non-responsive cultivar as a morphological/physiological counterpoint. Cultivar-specific agronomic backgrounds are noted in the Plant material section.

  1. Line 102. I recommend indicating the manufacturer of the growth chamber.

Responds: Thank you for the suggestion. We have added the manufacturer of the growth chamber: Yiheng, shanghai.

  1. Line 103. Please check the spelling of the degree Celsius symbol.

 

Responds: Corrected.

 

  1. Line 107. I recommend that you describe in more detail how you determined root activity and, if possible, provide a link to the reference. Please include the definition of TTC method.

Responds: We expanded the description to clarify that root activity was assayed via the triphenyl tetrazolium chloride (TTC) reduction method, with the reaction quantified spectrophotometrically. A citation has been added as follows:

Relative root activity was estimated using the triphenyl tetrazolium chloride (TTC) reduction method (Steponkus & Lanphear, 1967). Briefly, 0.5 g fresh root tips (taproots or adventitious roots) were incubated in 5 mL of 0.4% (w/v) TTC solution in 0.05 M phosphate buffer (pH 7.0) at 37 °C in darkness for 6 h. Formazan produced by mitochondrial dehydrogenase activity was extracted with 5 mL ethyl acetate by vigorous shaking, and the absorbance was measured at 485 nm using a spectrophotometer. Root activity was expressed as formazan content per gram fresh weight (mg g⁻¹ FW) or as relative activity normalized to the control.

  1. Line 111. "MTH-1". I recommend adding a definition of the abbreviation.

Responds: We have added the full definition of MTH-1: Manual Plant Microtome MTH-1.

  1. Line 113. I recommend indicating the manufacturer of the optical microscope.

Responds: We have added the manufacturer of the optical microscope information as follows:

The process of aerenchyma formation was observed under Olympus BX53 optical microscope (Olympus Corporation, Tokyo, Japan) equipped with a digital camera.

  1. Line 124. Why was transcriptional analysis only done on Zhonglü luffa cultivar?

Responds: We thank the reviewer for this question. RNA-seq was performed exclusively on ‘Zhonglü’ because this cultivar exhibited aerenchyma formation in the hypocotyl under waterlogging, whereas ‘Yalü’ was not  (see Figure 1). Since the primary aim of the transcriptomic assay was to capture the induced molecular program associated with aerenchyma initiation, sequencing a non-responding genotype would largely reflect housekeeping/stress-noise rather than aerenchyma-specific regulation. ‘Yalü’ was instead retained as a morphological and physiological contrast (root activity, anatomy) to demonstrate genotype-dependent sensitivity.

  1. Lines 129, 185, 224, 311. I recommend writing the p-value in lowercase letters.

Responds: Thank you for the suggestion. It has been revised.

  1. Line 142. I recommend indicating how your data are presented (means ±SD), how many independent experiments were conducted, and what p-value indicates significant differences (p<0.05). In the figure notes (Lines 183, 222, 309), I recommend indicating the statistical analysis method or test you used.

Responds: Thank you for the suggestion. It has been revised. We now explicitly state in the main text that data are presented as means ± SD, with sample sizes and replication noted, and that significance was set at p< 0.05. Each relevant figure note now specifies the statistical test used (e.g., Student’s t-test or two-way ANOVA with Tukey’s post hoc test).

Results

  1. Line 146. I recommend replacing “Morphological Changes” with “Morphological changes”.

Responds: Thank you for the suggestion. It has been revised.

  1. Line 159. ‘YL’ and ‘ZL’. I recommend adding definitions for these abbreviations. According to the journal rules, abbreviations should be expanded upon their first mention in a figure.

Responds: Thank you for the suggestion. It has been revised.

  1. Line 183. CK-ZL, WL-ZL, CK-YL, WL-YL. I recommend adding definitions for these abbreviations. In the figure note, I recommend indicating the statistical analysis method or test you used.

Responds: Thank you for the suggestion. It has been revised in the Figure 2 as follows:

No-waterlogging of Zhonglü (CK-ZL), Waterlogging of Zhonglü (WL-ZL), No-waterlogging of Yalü (CK-YL), Waterlogging of Yalü (WL-YL).

  1. Lines 202-205. I recommend editing or moving the discussion of the data from the results subchapter to the discussion chapter.

Responds: Thank you for the suggestion. Interpretive text originally in Lines 202–205 has been moved to the Discussion.

  1. Line 225. I recommend replacing “Effects of NPA and MeJA Application on aerenchyma formation Under waterlogging Stress” with “Effects of NPA and MeJA application on aerenchyma formation under waterlogging stress”.

Responds: Thank you for the suggestion. It has been revised.

  1. Line 294. Unfortunately, I could not find the definition of the TIFY9 gene in the manuscript. What is this gene, and what protein does it encode?

Responds: We apologize for the omission. A definition has been added as follows:

TIFY9 encodes a TIFY family transcription regulator containing the conserved TIF[F/Y]XG motif, involved in jasmonate signaling.

 

  1. Line 309. I recommend adding an explanation of the abbreviations for the genes being studied in the figure's note.

Responds: Thank you for the suggestion. Figure legend now lists full names of all studied genes. Gene abbreviations: ARF18, auxin response factor 18; IAA32, indole-3-acetic acid-induced protein 32; GH3, Gretchen Hagen 3 conjugating enzyme; TIFY9, jasmonate-associated TIFY transcription factor.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article is an attempt to study the genetic and physiological mechanisms of aerenchyma formation in the hypocotyl of luffa under waterlogging conditions.

The authors believe that auxin, methyl jasmonate, and the DEGs they identified are responsible for aerenchyma formation. However, in their experiments, luffa also exhibited a different response to flooding and these hormones—the formation of a mass of adventitious roots, from which lateral roots developed.

The reviewer is unclear why they concluded that these genes are responsible for aerenchyma development, rather than adventitious and lateral roots? This is especially true since the DEG list included genes involved in the auxin response, including orthologs of known root regulators.

In the reviewer's opinion, further experiments are required (not necessarily on transcriptome analysis, but at least on studying the influence of other hormones and using two varieties of luffa that contrast in their response to waterlogging).

 

Lines 16-18: differentially expressed genes (DEGs)associated with plant hormone signal transduction were implicated in this process, specifically highlighting fifteen auxin-related and two jasmonic acid-related genes.

I wouldn't be so categorical in claiming that genes associated with this process have been identified, especially in the abstract. Differential expression only suggests their involvement.

 

Lines 46-61 (About the formation of aerenchyma in roots)

From what tissue or tissues does aerenchyma originate? Only by line 257 does it become clear that in the luffa, this parent tissue is the bark. What about in other plants?

 

Lines 62-79 (Regarding the role of auxin and ethylene in aerenchyma development)

It is necessary to clarify whether they play a role in the development of the same type of aerenchyma tissue (lysigenic, schizogenic)? And is there any information about the mechanisms of their action in this process?

 

Lines 83-84: In this study, we 83 observed that the cultivar ‘Zhonglü’ developed aerenchyma in the hypocotyl under wa- 84 terlogging conditions, whereas ‘Yalü’ did not.

What type of aerenchyma does luffa form? Does it form only in the hypocotyls or in the roots as well?

 

Lines 165-181: Root activity measurement in ‘YL’ and ‘ZL’ following waterlogging stress

The term "Root activity" isn't very helpful. Based on the section's content, you might guess that it's probably about the rate of root growth. But that's not obvious, so please define it differently. Or, alternatively, at the beginning of the section (and in the caption to Figure 2), write what you mean by this term.

 

Lines 225-285 (Effects of NPA and MeJA Application on aerenchyma formation Under waterlogging Stress)

Why did you include these hormones in your study? Did other hormones influence aerenchyma formation (as well as the formation of adventitious roots and lateral roots on adventitious roots)?

Judging by the photographs, auxin and methyl jasmonate increased aerenchyma formation by 20 percent, and only under flooding. Moreover, only the 'ZL', which already forms aerenchyma and adventitious roots well, was studied.

The key role of these hormones in these processes would be worth discussing if they, and no other hormones, triggered the formation of adventitious roots and aerenchyma in the 'YL' variety as well. Or even without flooding.

I would suggest studying the effect of other phytohormones (ethylene? Gibberellins? Cytokinins?) on these processes, using the 'YL' variety alongside the 'ZL' variety. 

 

Lines 257-285 (Histological description of aerenchyma development in the loofah hypocotyl)

It's still unclear what type of aerenchyma loofah has, despite the photos provided. What happens during its formation: do the connections between cells weaken, or do some cells die? How might auxin and methyl jasmonate influence these processes? Is there any literature on this?

 

260-261: After 7 d and 14 d of waterlogging, the cortical cells of the hypocotyl elongated and extended to form aerenchyma.

It seems that during the formation of aerenchyma, cortical cells not only expand radially but also divide intensively: judging by the photo, their number increases dramatically. But there's no mention of this in the descriptions.

 

The Discussion rather lists the results obtained, but the Conclusion correctly states: “further research is needed.”

Comments for author File: Comments.pdf

Author Response

Responses to Reviewers’Comments

Referee: 3

The article is an attempt to study the genetic and physiological mechanisms of aerenchyma formation in the hypocotyl of luffa under waterlogging conditions.

 

The authors believe that auxin, methyl jasmonate, and the DEGs they identified are responsible for aerenchyma formation. However, in their experiments, luffa also exhibited a different response to flooding and these hormones—the formation of a mass of adventitious roots, from which lateral roots developed.

 

The reviewer is unclear why they concluded that these genes are responsible for aerenchyma development, rather than adventitious and lateral roots? This is especially true since the DEG list included genes involved in the auxin response, including orthologs of known root regulators.

In the reviewer's opinion, further experiments are required (not necessarily on transcriptome analysis, but at least on studying the influence of other hormones and using two varieties of luffa that contrast in their response to waterlogging).

Responds: We sincerely appreciate the reviewer’s critical and fair point. We fully agree that waterlogging induces a composite seedling response in luffa—hypocotyl aerenchyma co-occurring with profuse adventitious and lateral root emergence—and that transcriptomics cannot disentangle organ- or cell-type-specific regulation. Several auxin-related DEGs we highlighted (ARF18, IAA32, GH3) are indeed orthologs of known root developmental regulators, and their differential expression likely reflects this broader flooding syndrome rather than aerenchyma-exclusive control.

In the revised manuscript, we have removed language implying these genes are responsible for aerenchyma development. They are now presented strictly as candidate genes co-regulated during waterlogging, potentially linking polar auxin transport, jasmonate signaling, cortical remodeling, and adventitious root primordia activation. We explicitly acknowledge that future work should: (1) test additional phytohormones (ethylene, gibberellins, cytokinins) alongside auxin/MeJA, and (2) incorporate the non-responsive cultivar ‘Yalü’ into transcriptomic or spatial-expression analyses to better resolve genotype-dependent, organ-specific pathways. The reviewer’s constructive suggestion is now endorsed verbatim in the Discussion.

Lines 16-18: differentially expressed genes (DEGs) associated with plant hormone signal transduction were implicated in this process, specifically highlighting fifteen auxin-related and two jasmonic acid-related genes.

I wouldn't be so categorical in claiming that genes associated with this process have been identified, especially in the abstract. Differential expression only suggests their involvement.

Responds: We fully agree with the reviewer. Differential expression alone cannot establish causality, and we have accordingly softened the wording in the Abstract. The revised text no longer claims that genes “associated with this process have been identified,” the sentence has been revised as follows:

Transcriptome profiling of ‘Zhonglü’ revealed differentially expressed genes linked to plant hormone signal transduction, including fifteen auxin-related and two jasmonate-related genes, which may be involved in waterlogging-induced aerenchyma formation and are proposed as candidate regulators.

 

Lines 46-61 (About the formation of aerenchyma in roots)

From what tissue or tissues does aerenchyma originate? Only by line 257 does it become clear that in the luffa, this parent tissue is the bark. What about in other plants? ‘

Responds: We thank the reviewer for raising this point. We have revised this paragraph to explicitly state that aerenchyma most commonly originates from cortical parenchyma, clarify the distinction between root/stem and inner/outer cortical origins across species, and note that in herbaceous dicots (including Luffa) it develops in the outer cortex of the hypocotyl (sometimes loosely termed “bark” in non-woody axes). It has been revised as follows:

Aerenchyma is a specialized tissue in stems and roots containing air chambers that facilitate internal oxygen diffusion under flooding [15,16]. It most often originates from the cortical parenchyma, with developmental mode and parent layer varying among species. Two structural types are recognized: schizogenous, where cells separate without lysis, and lysigenous, where programmed cell death and cortical cell dissolution generate cavities [15,17,18]. Additionally, aerenchyma can be divided into two categories: inducible and constitutive. In upland crops (e.g., maize, wheat, barley, tomato), lysigenous aerenchyma is absent aerobically and induced only under oxygen deficiency [8,11,19–21]; in wetland species such as rice (Oryza sativa), lysigenous lacunae form constitutively in roots but are further enhanced by hypoxia [16,22]. In herbaceous dicots, including Luffa cylindrica, aerenchyma typically initiates in the outer cortex of the hypocotyl (the non-woody “bark” region beneath the epidermis), rather than from vascular or pith tissues, representing an inducible type that mitigates oxygen deficit. How this localized cortical acclimation is regulated under hypoxia remains poorly characterized and is the focus of the present study.

Lines 62-79 (Regarding the role of auxin and ethylene in aerenchyma development)

It is necessary to clarify whether they play a role in the development of the same type of aerenchyma tissue (lysigenic, schizogenic)? And is there any information about the mechanisms of their action in this process?

Responds: We thank the reviewer for this important clarification. The Introduction has been restructured to explicitly distinguish how auxin and ethylene act across lysigenous vs. schizogenous contexts. We now state that ethylene is best characterized in lysigenous root aerenchyma, where it induces ACS/ACO expression, ethylene burst, and ROS-mediated programmed cortical cell death (e.g., rice/maize), whereas auxin redistribution can influence both types: promoting lysigenous cavities via ethylene crosstalk or schizogenous spacing via cortical auxin maximum shifts and wall remodeling. The text now notes that in Luffa hypocotyls (predominantly expansigenous–lysigenous), aerenchyma arises in the outer cortex, and while direct cell-level evidence is limited, auxin–ethylene–ROS frameworks from cereals provide an interpretive basis. It has been revised as follows:

Several studies have demonstrated that aerenchyma formation is controlled by plant hormones. Ethylene is a primary driver of lysigenous aerenchyma in cereals: flooding-triggered ethylene accumulates via ACS/ACO induction, promoting ROS production and programmed cortical cell death, even under aerobic conditions with exogenous treatment [8,11,13,21,23–27]. Auxin modulates both root architecture and cortical remodeling; in rice, auxin signaling through AUX/IAA13–ARF pathways controls constitutive and hypoxia-enhanced lysigenous lacunae, with iaa13mutants suppressing apex aerenchyma [32,33]. In other contexts, auxin gradients or transport inhibition can generate schizogenous-like cavities by altering cortical cell separation and wall loosening [28–31]. Thus, auxin and ethylene can converge on the same tissue type (lysigenous) via ROS/cell-death modules, or act divergently depending on organ and species.

Lines 83-84: In this study, we observed that the cultivar ‘Zhonglü’ developed aerenchyma in the hypocotyl under waterlogging conditions, whereas ‘Yalü’ did not.

What type of aerenchyma does luffa form? Does it form only in the hypocotyls or in the roots as well?

 

Responds: We thank the reviewer for raising this. In Luffa cylindrica, waterlogging-induced aerenchyma in the hypocotyl develops in the outer cortex and has been described by Shimamura et al. as expansigeny—radial enlargement of cortical cells with subsequent cavity coalescence, showing lysigenous-like features at maturation. In this study, waterlogging induced aerenchyma in the outer cortex of ‘Zhonglü’ hypocotyls, a pattern consistent with expansigeny (radial cortical cell enlargement followed by lacunal coalescence; Shimamura et al., 2007). ‘Yalü’ showed negligible hypocotyl lacunae. Although luffa adventitious roots can also develop cortical air spaces (Shimamura et al., 2007), the taproot and basal root cortex in our 14-d flooding setup remained largely intact, with the dominant root response being adventitious root proliferation. We therefore focused anatomical and transcriptomic analyses on the hypocotyl, where aerenchyma was reproducible and quantifiable.

 

Lines 165-181: Root activity measurement in ‘YL’ and ‘ZL’ following waterlogging stress

The term "Root activity" isn't very helpful. Based on the section's content, you might guess that it's probably about the rate of root growth. But that's not obvious, so please define it differently. Or, alternatively, at the beginning of the section (and in the caption to Figure 2), write what you mean by this term.

Responds: We agree that “root activity” is ambiguous. The section heading and text have been revised to explicitly define this parameter as relative root metabolic activity assessed by the triphenyl tetrazolium chloride (TTC) reduction assay, which reflects mitochondrial dehydrogenase activity rather than root elongation rate. The Figure 2 caption has been updated accordingly.

Lines 225-285 (Effects of NPA and MeJA Application on aerenchyma formation Under waterlogging Stress)

Why did you include these hormones in your study? Did other hormones influence aerenchyma formation (as well as the formation of adventitious roots and lateral roots on adventitious roots)?

Judging by the photographs, auxin and methyl jasmonate increased aerenchyma formation by 20 percent, and only under flooding. Moreover, only the 'ZL', which already forms aerenchyma and adventitious roots well, was studied.

The key role of these hormones in these processes would be worth discussing if they, and no other hormones, triggered the formation of adventitious roots and aerenchyma in the 'YL' variety as well. Or even without flooding.

I would suggest studying the effect of other phytohormones (ethylene? Gibberellins? Cytokinins?) on these processes, using the 'YL' variety alongside the 'ZL' variety.

Responds: We appreciate the reviewer’s thoughtful critique. NPA and MeJA were selected because: (1) GO enrichment pointed to auxin and jasmonate signaling, and (2) prior studies link polar auxin transport and jasmonates to lysigenous aerenchyma or cortical cell death (e.g., in maize and Rumex). However, we fully agree that only ‘Zhonglü’ (the responsive cultivar) was tested, and the ~20% increase was flooding-dependent and modest in magnitude.

We have revised the text to state explicitly that NPA and MeJA are not claimed as exclusive or key triggers, but as modulators that accentuate a pre-existing, genotype-specific tendency. We now acknowledge that ethylene, gibberellins, and cytokinins were not examined, and that the contrasting silence of ‘Yalü’ suggests multiple hormonal layers. The Discussion now frames the hormone assays as hypothesis-generating and explicitly endorses the reviewer’s suggestion that multi-hormone, dual-cultivar screens are a logical next step.

It should be noted that NPA and MeJA modestly enhanced aerenchyma in ‘Zhonglü’ only under waterlogging, and were applied to a cultivar already predisposed to cortical remodeling. This does not exclude involvement of other phytohormones: ethylene, gibberellins, and cytokinins are well known to influence adventitious rooting and cortical plasticity, and may be particularly relevant to the non-responding ‘Yalü’. The present data therefore position auxin transport and jasmonate signaling as candidate modulatory nodes within a broader, genotype-dependent hormonal network, rather than sole determinants. Future work combining ‘Zhonglü’ and ‘Yalü’ with multi-hormone treatments will be required to resolve organ-specific regulatory hierarchies.

It has been revised in Discussion as follows:

It is important to emphasize that waterlogging induced a composite seedling syndrome in luffa, in which hypocotyl aerenchyma formation co-occurred with profuse adventitious and lateral root emergence. NPA and MeJA modestly enhanced aerenchyma in the responsive cultivar ‘Zhonglü’ only under concurrent flooding, and were tested here as exploratory modulators rather than universal triggers. It suggests that polar auxin transport and jasmonate signaling act as candidate nodes within a broader, genotype-dependent hormonal network. Other phytohormones—particularly ethylene, gibberellins, and cytokinins—are likely involved, especially given the muted cortical response of ‘Yalü’. Future comparative screens combining ‘Zhonglü’ and ‘Yalü’ with multi-hormone treatments and higher-resolution histology will be essential to disentangle organ-specific pathways.

 

Lines 257-285 (Histological description of aerenchyma development in the loofah hypocotyl)

It's still unclear what type of aerenchyma loofah has, despite the photos provided. What happens during its formation: do the connections between cells weaken, or do some cells die? How might auxin and methyl jasmonate influence these processes? Is there any literature on this?

Responds: We thank the reviewer for pushing us to clarify this. The revised text now states that in Luffa cylindrica hypocotyls, aerenchyma develops primarily through radial enlargement of outer cortical cells followed by intercellular space coalescence, a pattern Shimamura et al. termed expansigeny, intermediate between strict lysigenous and schizogenous forms. More recent anatomical work (Ferreira 2025) describes luffa cortical aerenchyma as schizo-lysigenous, with ROS-associated programmed cell death contributing to cavity maturation. We therefore describe our material as predominantly expansigenous with lysigenous-like features, rather than purely schizogenous.

Regarding hormonal influence: NPA (auxin transport blocker) is known to modulate auxin–ethylene crosstalk and can suppress or shift cortical PCD in other species (e.g., rice iaa13, NPA blocks aerenchyma; maize IAA+ACC promotes lysigenous cavities), while MeJA has been reported to amplify submergence-induced aerenchyma in maize roots via stress-primed cell clearance (Kawai et al. 2015). We cite these as analogous frameworks and explicitly note that direct cell-level evidence in luffa is lacking; our histological description is therefore confined to radial expansion, apparent periclinal divisions and cavity coalescence, with candidate roles for auxin transport and jasmonate left as correlative.

260-261: After 7 d and 14 d of waterlogging, the cortical cells of the hypocotyl elongated and extended to form aerenchyma.

It seems that during the formation of aerenchyma, cortical cells not only expand radially but also divide intensively: judging by the photo, their number increases dramatically. But there's no mention of this in the descriptions.

Responds: We thank the reviewer for this careful observation. The revised text now notes that, in addition to radial enlargement, cortical cells appear to increase in number during early aerenchyma development, suggestive of localized periclinal divisions in the outer cortex prior to cavity coalescence. It has been revised as follows:

Between 7 and 14 d of waterlogging, cortical cells in the outer hypocotyl expanded radially and, in places, appeared to increase in number, suggesting localized periclinal divisions before the coalescence of intercellular spaces into aerenchyma cavities.

 

The Discussion rather lists the results obtained, but the Conclusion correctly states: “further research is needed.”

Responds: We sincerely thank the reviewer for this constructive comment. We agree that the original Discussion placed too much emphasis on result description. We have therefore substantially reorganized the Discussion to move beyond a simple summary of findings. It has been revised in the manuscript.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, thank you for editing the manuscript in accordance with my recommendations.

Lines 106-107. Please specify the camera model. For example, the LRH-250CB growth camera (Yiheng, Shanghai, China).

I have no further comments. I wish you success in your future research.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors of the article have made edits to the manuscript and responded to the reviewer's comments. In my opinion, the article can be accepted for publication in its current form.