Review Reports - Potassium Stress Induces Compensatory Root Adaptive Responses in Trifoliate Orange Through Reconfigured Auxin Signaling

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Based on my evaluation, the study by Chun-Yan Liu et al. (2026), in the manuscript entitled “Potassium stress induces compensatory root developmental responses in trifoliate orange through reconfigured auxin signaling”, addresses a relevant topic and presents a substantial amount of experimental data, particularly in a woody perennial system, which is of interest to the journal’s readership. However, after carefully evaluating the manuscript, I believe that it currently requires major revisions before it can adequately support the conclusions and the conceptual framework proposed by the authors.

In my view, several aspects would benefit from significant improvement, including:

clearer articulation of the central message and the working hypothesis,
stronger support for the proposed compensatory interpretation of root hair responses,
improved clarity, representativeness, and description of key images; particularly those related to root hair traits, and
additional experimental validation and/or a more cautious interpretation of the existing data.

Furthermore, the description and interpretation of the schematic model and several figures would benefit from greater detail, in order to improve clarity and consistency with the results presented.

While the manuscript has potential, substantial revisions are needed to improve the clarity of the message, the quality of the visual data, and the strength of the experimental and interpretative support. For these reasons, I would recommend the option of major revision.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Author Response

Comment 1: Chun-Yan Liu et al. 2026, in the manuscript entitled “Potassium stress induces compensatory root developmental responses in trifoliate orange through reconfigured auxin signaling”, study how different potassium (K⁺) levels affect plant growth and root system architecture in Poncirus trifoliata seedlings, with special attention to hormonal responses and gene expression. The authors evaluate plant performance under four contrasting external K⁺ concentrations (low, medium, control, and high), analysing shoot and root growth, root architectural traits, and root hair development. In parallel, they quantify endogenous levels of several phytohormones and analyse the expression of genes related to expansins as well as auxin biosynthesis and transport.

From these data, the authors propose that both K deficiency and potassium excess inhibits overall root growth when compared with the sufficient condition, and that this inhibition is accompanied by compensatory developmental responses, mainly reflected as increased root hair density, length and thickness. These responses are interpreted as adaptive adjustments to mantain root function under K stress and are discussed in relation to changes in hormone balance and in gene expression patterns associated with auxin- and expansin-related pathways. Based on this interpretation, the authors propose an integrative model linking K availability, hormone signaling and root developmental plasticity.

The manuscript highlights the relevance of this work by focusing on trifoliate orange as an important citrus rootstock and as a representative woody perennial species. In this context, the authors suggest that the observed root responses to K availability could be relevant for other related plant groups. This perspective adds value to the study, as responses of woody perennials to K availability are still less explored that in herbaceous model species.

Response: We sincerely thank the Reviewer for recognizing the value of our work, particularly our focus on the woody perennial Poncirus trifoliata. We appreciate the positive assessment regarding the relevance of our study to the broader field of plant nutrition and root development in non-model species. We have carefully addressed the specific concerns raised in the subsequent comments to strengthen the support for our proposed framework and improve the manuscript’s clarity..

Comments 2: Major observations

While the manuscript presents a large amount of descriptive data generated with clear experimental effort, several points emerged during the reading that, in my opinion, limit how clearly the proposed compensatory framework is supported by the results presented.

One central point is that, although the manuscript suggests that inhibition of general root growth under K deficiency or excess leads to compensatory enhancement of root hair traits, this idea is not always clearly stated nor consistently supported across the different datasets. In some parts, reduced root growth and increased root hair development are described in parallel, but the functional connection remains mostly inferred.

Response: We appreciate the Reviewer for pointing out this gap in our narrative. We agree that while we observed a clear “morphological dichotomy” (inhibited architecture vs. enhanced root hairs), the functional link between them was primarily interpreted through the lens of resource allocation theory rather than direct functional assays.

To clarify this compensatory framework, we have revised Discussion Section 4.2 to explicitly frame these responses as a metabolic trade-off. We now elaborate on the concept that under K⁺ stress (which limits photosynthesis and turgor), the plant strategically shifts carbon allocation from “expensive” macro-architectural growth (lateral roots) to “low-cost” micro-architectural expansion (root hairs) to maintain absorptive surface area. Furthermore, as noted in our response to Reviewer 1, we have added a Limitation Statement in Section 4.6 to acknowledge that functional verification of this compensation (e.g., K uptake efficiency assays) is a subject for future research.

Comment 3: This issue is closely related to the lack of direct measurements of internal K status. The interpretation of potassium deficiency, sufficiency and excess is based only on the external K concentrations applied. No measurements of K content in roots or shoots, nor the use of known physiological or molecular markers of K status, are presented. Because of this, it is difficult to be sure that the treatments generated clearly distinct internal K conditions, which weakens the interpretation of root hair responses as true compensatory adaptations.

Response: We acknowledge this as a significant limitation in our experimental characterization. While we did not directly quantify internal K⁺ content or physiological markers in this specific study, the experimental concentrations (0, 2, 6, 12 mmol/L) were selected based on established protocols for trifoliate orange (Cao et al., 2014) which have been previously shown to induce distinct nutritional states.

Furthermore, the plants exhibited clear and significant morphological divergence consistent with K⁺ stress gradients: the severe biomass reduction (Table 2) and distinct root architectural changes (Figure 2) observed under K₀ and K₁₂ treatments provide strong biological evidence that the external treatments successfully imposed varying levels of physiological stress.

Comment 4: Lack of Functional Evidence

In addition, although increased root hair density and length are presented as compensatory traits, the manuscript do not provide functional evidence showing that these changes improve K uptake or plant performance. Measurements related to K uptake efficiency or nutrient acquisition would help to better support this interpretation.

Response: We accept this constructive criticism. We agree that our study infers the “compensatory” function of root hairs based on morphological geometry (increased surface area) rather than direct physiological measurements of K⁺ uptake rates.

While conducting these uptake kinetics experiments was beyond the scope of the current morphological and molecular study, we have updated the “Future Directions” in Discussion Section 4.6. We now explicitly list “quantifying K⁺ uptake efficiency” as a priority for follow-up research to functional validate whether the observed root hair proliferation translates into improved nutrient acquisition status.

Comment 5: The hormonal and gene expression analyses provide useful information, but they remain largely correlational. In several parts of the discussion, changes in hormone levels and gene expression are interpreted as drivers of the observed root responses... This point is especially important for auxin, since its developmental effects depend strongly on spatial distribution rather than only on total hormone levels.

Response: We completely agree with the Reviewer. We recognize that total root hormone levels (measured via ELISA) do not capture the fine-scale spatial gradients of auxin that ultimately regulate root patterning.

However, to address the importance of spatial distribution, our study included a comprehensive expression analysis of auxin transporter genes (PtPINs, PtAUX1, PtLAXs, PtABCBs) (Figure 8). The significant upregulation of efflux carriers (e.g., PtPIN1, PtPIN4) and influx carriers under K⁺ stress provides indirect molecular evidence that the plant is actively reconfiguring auxin transport machinery to alter spatial gradients, even if we could not visualize these gradients directly.

To address the concern about “correlation vs. causality”, we have extensively revised the manuscript (as detailed in our response to Reviewer 1). We have:

Toned down the language: Replaced strong causal verbs (e.g., “drive”, “determine”) with interpretative terms (e.g., “suggest”, “is associated with”) throughout the Abstract and Discussion.

Added a Limitation Statement: Explicitly acknowledged in Discussion Section 4.6 that our proposed mechanisms are based on transcriptomic correlations and that causal links remain to be verified.

Comment 6: Another point concerns the root hair images (Figure 3). The micrographs show a high level of variability in root hair density, length and thickness, which makes it difficult to visually connect them with the quantitative values shown in the graphs. Clearer and more representative images... would strengthen the support.

Response: We thank the Reviewer for this observation. We acknowledge that biological variability in root hair development can make individual micrographs appear heterogeneous.

We wish to clarify that the quantitative data presented in Figure 4 was derived from a rigorous analysis of 24 independent root segments per treatment (as detailed in Materials and Methods Section 2.3), rather than relying solely on the specific fields of view shown in Figure 3. The SEM images in Figure 3 were selected to represent the general morphological trends (e.g., the presence/absence of density changes) rather than to serve as the sole basis for quantification.

To address the Reviewer’s concern about connecting images to data, we have revised the Legend of Figure 3. We added a statement explicitly clarifying that these micrographs are representative examples and that the quantitative values in Figure 4 represent the statistical average of the larger dataset, which accounts for the observed natural variability.

Comment 7: The schematic model presented in the last figure (Figure 11) is helpful... but its description is too general. The legend does not clearly explain how each element of the model is supported by the experimental data.

Response: We thank the Reviewer for this suggestion. We agree that the original legend was overly brief and did not adequately guide the reader through the proposed mechanistic pathways.

To improve clarity, we have completely rewritten the Legend of Figure 11. The revised caption now explicitly narrates the sequence of events supported by our data: it details how K⁺ stress leads to specific hormonal shifts (reduced IAA/GAs/ZR) and the subsequent transcriptional reconfiguration of auxin-related (PtTAA/YUC/PIN) and expansin (PtEXPA) genes, which collectively drive the compensatory root hair development.

Comment 8: The manuscript presents an interesting idea; that inhibition of root growth under K stress is accompanied by compensatory enhancement of root hair development, but this central concept would benefit from clearer explanation and stronger experimental support. Addressing the points mentioned above would improve the coherence of the proposed framework and increase confidence in the authors conclusions.

Response: We sincerely thank the Reviewer for this encouraging summary and for the constructive guidance provided throughout the review process.

In addition, the figures and tables are improved as requested, The language was also polished by professional Jiadong He, who is conducting research on plant nutrition at the University of Leuven in Belgium.

Reviewer 2 Report

Comments and Suggestions for Authors

Major Strengths

Integrated Multiscale Approach
The study successfully integrates morphological, physiological, and molecular analyses, combining root architecture traits, root hair phenotypes, endogenous hormone measurements, and gene expression profiling. This comprehensive design strengthens the biological interpretation of potassium-induced plasticity.
Clear Identification of Optimal and Stress K⁺ Levels
The identification of K6 (6 mmol L⁻¹) as an optimal concentration for root architectural development is supported by consistent morphological data and reinforced through principal component analysis (PCA), which explains over 81% of cumulative variance using three principal components.
Detailed Root Phenotyping
High-resolution root system analysis (WinRHIZO) and scanning electron microscopy-based root hair characterization provide robust quantitative and qualitative evidence of distinct architectural versus root hair responses under potassium stress.
Hormonal and Gene Expression Correlation Framework
The correlation analyses linking auxin (IAA), gibberellins (GAs), and zeatin riboside (ZR) with root traits, as well as expansin and auxin-transport gene expression with root hair phenotypes, offer a plausible mechanistic framework for compensatory developmental responses.

Major Limitations and Methodological Concerns

Causality versus Correlation
While strong correlations between hormone levels, gene expression, and root traits are reported, the study remains largely correlative. The interpretation that auxin reconfiguration drives compensatory root hair development is plausible but not experimentally demonstrated. Functional validation is lacking.
Limited Temporal Resolution
All measurements were taken after 10 weeks of K⁺ treatment. This single end-point limits insight into the temporal dynamics of hormonal signaling and gene regulation. Early versus late responses to potassium stress cannot be distinguished.
Restricted Hormone Quantification Methodology
Hormone levels were measured using ELISA, which, while acceptable, may lack specificity and sensitivity compared to LC–MS/MS. Cross-reactivity and absolute quantification accuracy should be acknowledged as a limitation.
Single Genotype and Controlled Conditions
The exclusive use of trifoliate orange seedlings grown under controlled conditions limits the generalizability of the findings. Genotypic variation and soil-based or field conditions may yield different adaptive responses.
Interpretation of PCA-Derived “Optimal K⁺”
Although PCA is appropriately applied, the interpretation of K6 as the “optimal” concentration depends on the selected traits and their weighting. The term “optimal” should be contextualized as optimal within the measured parameter space and experimental conditions.

Author Response

Comment 1: Major Strengths

Integrated Multiscale Approach

The study successfully integrates morphological, physiological, and molecular analyses, combining root architecture traits, root hair phenotypes, endogenous hormone measurements, and gene expression profiling. This comprehensive design strengthens the biological interpretation of potassium-induced plasticity.

Clear Identification of Optimal and Stress K⁺ Levels

The identification of K6 (6 mmol L⁻¹) as an optimal concentration for root architectural development is supported by consistent morphological data and reinforced through principal component analysis (PCA), which explains over 81% of cumulative variance using three principal components.

Detailed Root Phenotyping

High-resolution root system analysis (WinRHIZO) and scanning electron microscopy-based root hair characterization provide robust quantitative and qualitative evidence of distinct architectural versus root hair responses under potassium stress.

Hormonal and Gene Expression Correlation Framework

The correlation analyses linking auxin (IAA), gibberellins (GAs), and zeatin riboside (ZR) with root traits, as well as expansin and auxin-transport gene expression with root hair phenotypes, offer a plausible mechanistic framework for compensatory developmental responses.

Response: We sincerely thank the Reviewer for this positive assessment and the accurate summary of our work. We appreciate the recognition of our detailed root phenotyping using WinRHIZO and SEM, as well as the correlation framework established between hormonal changes and gene expression.

Comment 2: Major Limitations and Methodological Concerns

Causality versus Correlation

While strong correlations between hormone levels, gene expression, and root traits are reported, the study remains largely correlative. The interpretation that auxin reconfiguration drives compensatory root hair development is plausible but not experimentally demonstrated. Functional validation is lacking.

Response: We sincerely thank the Reviewer for this critical and insightful comment. We fully agree that while our integrated analysis reveals strong associations between gene expression, hormone profiles, and root phenotypes, our current data is correlative and does not definitively prove causality.

To address this concern rigorously, we have made the following revisions:

Toned down the language: We have carefully reviewed the entire manuscript (Abstract, Discussion, and Conclusions) and replaced strong causal verbs (e.g., “demonstrate”, “establish”, “confirm”) with more interpretative terms (e.g., “suggest”, “indicate”, “support”) when describing molecular mechanisms.
Added a Limitation Statement: We have restructured the final paragraph of Discussion Section 4.6. We now explicitly acknowledge the limitations of our study design at the beginning of the concluding remarks, prior to discussing future directions. Specifically, we state that “causal links remain to be experimentally verified” and propose specific genetic approaches (e.g., CRISPR/Cas9) as a priority for future research.

Comment 3: Limited Temporal Resolution

All measurements were taken after 10 weeks of K⁺ treatment. This single end-point limits insight into the temporal dynamics of hormonal signaling and gene regulation. Early versus late responses to potassium stress cannot be distinguished.

Response: We appreciate this valid observation. We acknowledge that a single sampling point prevents distinguishing between early signaling and steady-state maintenance.

As part of the restructuring of Discussion Section 4.6 (mentioned in the response to Comment 2), we have placed the limitation regarding the single time-point at the forefront of our concluding remarks. We clarify that while the 10-week point captures long-term traits relevant to woody perennials, future time-course analyses are needed to resolve dynamic changes.

Comment 4: Restricted Hormone Quantification Methodology

Hormone levels were measured using ELISA, which, while acceptable, may lack specificity and sensitivity compared to LC–MS/MS. Cross-reactivity and absolute quantification accuracy should be acknowledged as a limitation.

Response: We fully agree with the Reviewer regarding the limitations of the ELISA methodology. While ELISA is a widely accepted, high-throughput method suitable for detecting relative trends and treatment effects in plant physiology, we acknowledge that LC-MS/MS offers superior specificity and sensitivity, particularly regarding absolute quantification and minimizing cross-reactivity.

In line with the restructuring of our discussion on limitations (as detailed in previous responses), we have integrated a specific acknowledgement of this methodological constraint. We explicitly state that while our ELISA results are valid for comparing relative differences, they should be interpreted with awareness of potential cross-reactivity, and we have added a recommendation for LC-MS/MS validation in future studies.

In the restructured Section 4.6, we have added a second point to the limitations paragraph: “Second, endogenous hormones were quantified using ELISA. While this method effectively highlights relative differences between treatments, we acknowledge that it generally lacks the absolute specificity and sensitivity of LC-MS/MS due to potential antibody cross-reactivity”. We also updated the future directions to include “verifying hormonal profiles using LC-MS/MS”.

Comment 5 Single Genotype and Controlled Conditions

The exclusive use of trifoliate orange seedlings grown under controlled conditions limits the generalizability of the findings. Genotypic variation and soil-based or field conditions may yield different adaptive responses.

Interpretation of PCA-Derived “Optimal K⁺”

Although PCA is appropriately applied, the interpretation of K6 as the “optimal” concentration depends on the selected traits and their weighting. The term “optimal” should be contextualized as optimal within the measured parameter space and experimental conditions.

Response: We thank the Reviewer for these precise and constructive comments regarding generalizability and data interpretation.

Regarding Genotype and Conditions: We agree that findings from a single genotype in a controlled environment may not fully represent field scenarios or inter-specific variations. In the restructured limitations paragraph (Discussion Section 4.6), we have added a specific point acknowledging this limitation. We have also updated our future directions to explicitly call for “multi-genotype field trials” to validate these adaptive responses in complex soil environments.

Regarding PCA Interpretation: We agree that “optimal” is a relative term dependent on the traits weighted in the PCA. We have revised the relevant text in the Results section (Section 3.8) to contextualize our conclusion. We now specify that K₆ is optimal specifically “for the measured root system architecture traits under these experimental conditions”, avoiding overgeneralization to total yield or field survival.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate the effort invested in revising the manuscript and adjusting the interpretation of the results.

I still have some concerns regarding the use of the term “compensatory”. Based on the data presented, it is not fully clear whether the increase in root hair density, length, thickness, and morphology represents a compensatory response to the inhibition of primary and lateral root growth under K deficiency or excess. The study provides descriptive, hormonal, and transcriptional information, but as I commented before it does not include experimental evidence showing that root hair proliferation restores or maintains nutrient acquisition or plant performance under these conditions. I suggest reconsidering the use of the term “compensatory”, or at least clarifying that this interpretation remains hypothetical, and would require functional validation.

Another minor point; the terminology used to describe the K6 treatment would benefit from greater consistency throughout the manuscript. The text alternates between terms such as optimal and moderate. It would help if the authors select one term and use it consistently.

The discussion could benefit from including additional literature on root architectural plasticity and carbon allocation under nutrient stress, such as the conceptual framework proposed by: (https://doi.org/10.1093/aob/mcs293), which is relevant for understanding resource optimization and root architectural adjustments based on soil mineral conditions,.

Best regards,

Author Response

Comment 1: I still have some concerns regarding the use of the term “compensatory”. The study provides descriptive, hormonal, and transcriptional information, but. it does not include experimental evidence showing that root hair proliferation restores or maintains nutrient acquisition or plant performance. I suggest reconsidering the use of the term “compensatory”, or at least clarifying that this interpretation remains hypothetical.

Response: We agree with the reviewer that our current data (morphological, hormonal, and transcriptomic) describes a strong adaptive response but does not provide direct physiological evidence (such as K⁺ uptake kinetics) to prove that root hair proliferation fully “compensates” for the inhibited root system in terms of nutrient acquisition.

To address this, we have revised the title and the text to use broader terms such as “adaptive” or “plasticity” instead of “compensatory” where appropriate.

In the Discussion section (specifically Section 4.2.2), we have explicitly clarified that while the observed root hair proliferation is likely a strategy to maintain absorption surface area, its function in restoring plant performance remains hypothetical in this study and requires further functional validation (e.g., K⁺ uptake efficiency quantification).

Comment 2: The terminology used to describe the K₆ treatment would benefit from greater consistency throughout the manuscript. The text alternates between terms such as optimal and moderate.

Response: Thank you for pointing out this inconsistency. We have standardized the terminology throughout the manuscript. We now consistently use “moderate” to describe the K⁺ concentration level (i.e., “Moderate K” or “Moderate K treatment”) and reserve the word “optimal” only when discussing the resultant growth performance (e.g., “seedlings under moderate K treatment exhibited optimal root architecture”).

Comment 3: The discussion could benefit from including additional literature on root architectural plasticity and carbon allocation under nutrient stress, such as the conceptual framework proposed by: (https://doi.org/10.1093/aob/mcs293).

Response: We appreciate this valuable reference. We have incorporated the work of Postma et al. (2014) into the Discussion section (Section 4.2.1). We used this reference to strengthen our discussion regarding the trade-offs between the metabolic costs of root construction and resource acquisition strategies under nutrient stress.