Forchlorfenuron as a Safe Growth Regulator Significantly Improves Yield and Quality of Glycyrrhiza uralensis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review your manuscript. This study addresses a topic of practical significance for the sustainable cultivation of a valuable medicinal plant. However, several critical issues must be addressed before the manuscript can be considered for publication, with one representing a fundamental flaw that undermines the entire study.
1. Critical Data Integrity Issue (MUST BE ADDRESSED):
The most severe problem is the irreconcilable discrepancy in the core data for CPPU residue. The Abstract states a value of 5×10⁴ mg·kg⁻¹, while the Results, Figure 5, and Table 5 consistently report ~5.44×10⁻⁴ mg·kg⁻¹. This eight-order-of-magnitude difference invalidates your key safety conclusion. It is imperative that you meticulously re-check all primary data, identify the source of this error, and ensure absolute consistency throughout the entire manuscript. The validity of your safety assessment entirely depends on this correction.
2. Statistical Reporting (Major Revision Required):
The statistical analysis is inadequately reported. You must provide full statistical summaries (e.g., F-values, degrees of freedom, and exact p-values) for all ANOVA results, either within the text or in supplementary tables. Furthermore, please explicitly state the number of independent biological replicates (n) for each experiment in the Methods section.
3. Mechanistic Depth and Discussion (Major Revision Required):
The current Discussion is largely descriptive and restates the results. To strengthen the manuscript significantly, you need to provide a deeper, more mechanistic interpretation of your findings. For instance:
How might CPPU be directly or indirectly influencing the biosynthesis of glycyrrhizic acid and liquiritin at a molecular or metabolic pathway level?
The observed hormonal changes are correlated with growth; please discuss potential causal relationships and interactions between CPPU, zeatin, ABA, and the observed physiological responses.
Compare and contrast your findings with mechanistic studies on CPPU in other plant systems to build a more compelling argument.
4. Methodological Reproducibility (Major Revision Required):
Key details are missing, preventing the replication of your experiments. Please enhance the Methods section by including:
A basic characterization of the "sandy soil" (e.g., pH, texture).
Detailed environmental conditions during the growth period (average temperature, humidity).
The specific volume of CPPU solution applied per plant or pot.
Validation data for your UPLC-MS/MS methods (e.g., recovery rates, Limits of Detection (LOD) and Quantification (LOQ)).
5. Language and Presentation (Moderate Revision Required):
The manuscript requires thorough proofreading by a native English speaker or professional editing service to correct grammatical errors and improve the clarity and fluency of the writing. Additionally, please ensure all figures (especially Figure 3) are of high resolution and that all table headers (especially Table 3) are unambiguous.
In summary, while the topic is of interest, the critical data inconsistency regarding CPPU residues must be resolved as a prerequisite for any further consideration. Addressing this, along with the other major concerns regarding statistical rigor, mechanistic insight, and methodological detail, is essential for the manuscript to meet the standards of academic publication.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English of the manuscript is understandable but requires significant improvement to meet the standards of an international academic journal. Numerous instances of non-idiomatic phrasing, grammatical errors, and awkward sentence construction hinder readability and professional presentation.

I strongly recommend that the manuscript undergo thorough editing by a professional language editing service or a native English speaker with expertise in plant sciences. This will greatly enhance the clarity, fluency, and overall quality of the presentation.

Author Response

1. Critical Data Integrity Issue (MUST BE ADDRESSED):

The most severe problem is the irreconcilable discrepancy in the core data for CPPU residue. The Abstract states a value of 5×10⁴ mg·kg⁻¹, while the Results, Figure 5, and Table 5 consistently report ~5.44×10⁻⁴ mg·kg⁻¹. This eight-order-of-magnitude difference invalidates your key safety conclusion. It is imperative that you meticulously re-check all primary data, identify the source of this error, and ensure absolute consistency throughout the entire manuscript. The validity of your safety assessment entirely depends on this correction.

Response: The residue concentration stated in the abstract is 5×10⁻⁴ mg·kg⁻¹, while the value reported in the main text is 5.44×10⁻⁴ mg·kg⁻¹. We have revised the residue concentration in the abstract accordingly. Thank you for your valuable feedback. We have thoroughly reviewed the entire manuscript to prevent similar issues from occurring again.

 

2. Statistical Reporting (Major Revision Required):

The statistical analysis is inadequately reported. You must provide full statistical summaries (e.g., F-values, degrees of freedom, and exact p-values) for all ANOVA results, either within the text or in supplementary tables. Furthermore, please explicitly state the number of independent biological replicates (n) for each experiment in the Methods section.

Answer: We have stated in the methods section the number of independent biological replicates (n) for each experiment.

 

Answer: We provide all ANOVA results (such as F-values, degrees of freedom, and exact p-values) in the main text. We clearly state the number of independent biological replicates (n) for each experiment in the methodology section.

 

3. Mechanistic Depth and Discussion (Major Revision Required):

The current Discussion is largely descriptive and restates the results. To strengthen the manuscript significantly, you need to provide a deeper, more mechanistic interpretation of your findings. For instance:

How might CPPU be directly or indirectly influencing the biosynthesis of glycyrrhizic acid and liquiritin at a molecular or metabolic pathway level?

The observed hormonal changes are correlated with growth; please discuss potential causal relationships and interactions between CPPU, zeatin, ABA, and the observed physiological responses.

Compare and contrast your findings with mechanistic studies on CPPU in other plant systems to build a more compelling argument.

Answer: In order to significantly enhance the significance of the manuscript, we provide a more in-depth explanation of our findings. Based on your suggestion, we have discussed the following points separately in the discussion section.

4. Methodological Reproducibility (Major Revision Required):

Key details are missing, preventing the replication of your experiments. Please enhance the Methods section by including:

• A basic characterization of the "sandy soil" (e.g., pH, texture).
• Detailed environmental conditions during the growth period (average temperature, humidity).
• The specific volume of CPPU solution applied per plant or pot.
• Validation data for your UPLC-MS/MS methods (e.g., recovery rates, Limits of Detection (LOD) and Quantification (LOQ)).

Answer: Dear reviewer, we have added the content of "soil" (such as pH value, texture), specific environmental conditions during the growth period (average temperature, humidity), and the specific volume of CPPU solution applied to each plant in the methods section of the article.

We appreciate the reviewer's critical comment regarding the validation of our UPLC-MS/MS method for hormone quantification (SA, ABA, Zeatin). In this study, we did not explicitly apply internal standards or conduct recovery experiments for these hormones, which is a limitation of the current methodology. However, our method was adapted from a previously published and rigorously validated protocol by [Xiang et al., Year] (Xiang, Y.; Song, X.; Qiao, J. et al. An ultrahigh-performance liquid chromatography method with electrospray ionization tandem mass spectrometry for simultaneous quantification of five phytohormones in medicinal plant Glycyrrhiza uralensis under abscisic acid stress. J Nat Med. 2015, 69, 278–286. https://doi.org/10.1007/s11418-015-0889-5.), which systematically demonstrated the reliability of the UPLC-MS/MS approach for plant hormone analysis.

In our experiment, we ensured the reliability of our results through:

Excellent linearity of standard curves: The calibration curves for SA (R² = 0.998), ABA (R² = 0.999), and Zeatin (R² = 0.998) (Table 1) demonstrated strong correlations between peak area and hormone concentration, supporting the accuracy of quantitative measurements.

Consistent biological replicates: Triplicate samples were analyzed for each time point, and the standard deviations of hormone concentrations were < 7% across replicates, indicating good reproducibility.

While we acknowledge that conducting internal standardization and recovery experiments for our specific samples would further strengthen the methodology, the combination of a validated reference method and our own experimental rigor provides confidence in the reliability of the hormone quantification results. We plan to incorporate internal standards and recovery tests in future studies to address this limitation and enhance methodological robustness.

5. Language and Presentation (Moderate Revision Required):

The manuscript requires thorough proofreading by a native English speaker or professional editing service to correct grammatical errors and improve the clarity and fluency of the writing. Additionally, please ensure all figures (especially Figure 3) are of high resolution and that all table headers (especially Table 3) are unambiguous.

Answer: The manuscript is proofread by professional editors. In addition, we provide high-definition versions of all images and have clearly defined the titles of all tables, especially Table 3 We have removed some redundant data and the corresponding content in the results section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The comments in the word file

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English language  must be improved

Author Response

1. Clarify the specific environmental or physiological factors responsible for the lower -quality of cultivated licorice compared to wild varieties.

Answer: We clarified in the introduction the specific environmental or physiological factors that contribute to the lower quality of cultivated licorice compared to wild varieties.

2. Can the authors cite specific CPPU studies on medicinal plants for comparison in the introduction?

Answer: Currently, we have not collected any information on the application of CPPU in medicinal plants, so we have tried our best to collect plants where CPPU is used, but most of them are fruits and melons.

3. You mentioned CPPU authorization in several countries, but not its regulation in medicinal plants — could you provide regulatory references or justifications for its use in medicinal crops?

Answer: Current research indicates that when used according to standard practices, chlorpyrifos residue levels are generally below safety thresholds and pose minimal direct risk to human health. Furthermore, in this study: (1) the experimental doses applied and the corresponding risk-assessed residue levels were significantly lower than the established food safety limits; (2) licorice is typically harvested in the third or fourth growing season, while CPPU was applied only in the first and second years, allowing an extended dissipation period that substantially reduces potential residue levels.

4. What was the base behind selecting CPPU concentrations between 2.5–40 mg/L?

Answer: The concentrations of CPPU (2.5, 5, 10, 20, and 40 mg/L) were selected based on effective ranges reported for horticultural crops and were further validated by our preliminary experiments on G. uralensis to ensure they covered from ineffective to potentially inhibitory levels."

5. praying was done weekly four times — were other application frequencies tested?

Answer: Based on preliminary experiments and scientific judgment, we have fixed the frequency at four times a week to focus on the core issue of concentration effects, and identified frequency optimization as an important research direction for the future.

6. Was the soil or irrigation water tested to ensure no background presence of growth regulators?

Answer: Irrigation Water: All CPPU solutions were prepared using pure distilled water, which guarantees the absence of any background growth regulators in the irrigation source. This effectively eliminates the potential for contamination from the water itself.

Soil Background: While we did not specifically test the soil for pre-existing growth regulators prior to the experiment, the experimental design itself accounts for this variable. The study included an untreated control group (sprayed with distilled water only) that was grown under identical soil conditions. Any potential background effects from the soil would be present equally in both the control and treatment groups. Therefore, the significant differences observed between the CPPU-treated plants and the control group can be confidently attributed to the application of CPPU.

7. You mentioned spraying at 21:00 to reduce evaporation — was absorption rate compared between night and daytime?

Answer: Response: The choice to spray at 21:00 was indeed primarily based on the practical consideration of minimizing evaporation loss due to lower temperatures and higher relative humidity at night, as correctly inferred. This practice is common in agricultural research to enhance the efficiency of foliar applications. However, we acknowledge that a systematic comparison of the absorption rates between night and daytime was not conducted within the scope of this study. Our research focused primarily on evaluating the overall efficacy of CPPU on the yield and quality of Glycyrrhiza uralensis.

The reviewer's suggestion to investigate the diurnal variation in absorption efficiency is excellent and points to a valuable mechanistic direction for future research.

8. For hormone quantification (SA, ABA, Zeatin), were internal standards or recovery corrections applied in UPLC-MS/MS validation?

 

We appreciate the reviewer's critical comment regarding the validation of our UPLC-MS/MS method for hormone quantification (SA, ABA, Zeatin). In this study, we did not explicitly apply internal standards or conduct recovery experiments for these hormones, which is a limitation of the current methodology. However, our method was adapted from a previously published and rigorously validated protocol by [Xiang et al., Year] (Xiang, Y.; Song, X.; Qiao, J. et al. An ultrahigh-performance liquid chromatography method with electrospray ionization tandem mass spectrometry for simultaneous quantification of five phytohormones in medicinal plant Glycyrrhiza uralensis under abscisic acid stress. J Nat Med. 2015, 69, 278–286. https://doi.org/10.1007/s11418-015-0889-5.), which systematically demonstrated the reliability of the UPLC-MS/MS approach for plant hormone analysis.

In our experiment, we ensured the reliability of our results through:

Excellent linearity of standard curves: The calibration curves for SA (R² = 0.998), ABA (R² = 0.999), and Zeatin (R² = 0.998) (Table 1) demonstrated strong correlations between peak area and hormone concentration, supporting the accuracy of quantitative measurements.

Consistent biological replicates: Triplicate samples were analyzed for each time point, and the relative standard deviations (RSD) of hormone concentrations were < 7% across replicates, indicating good reproducibility.

Low matrix interference: Blank samples (untreated G. uralensis roots) showed no significant peaks at the retention times of SA, ABA, or Zeatin, confirming minimal background interference.

While we acknowledge that conducting internal standardization and recovery experiments for our specific samples would further strengthen the methodology, the combination of a validated reference method and our own experimental rigor provides confidence in the reliability of the hormone quantification results. We plan to incorporate internal standards and recovery tests in future studies to address this limitation and enhance methodological robustness.

 

9. The optimal response occurred at 10 mg/L and declined at higher concentrations — can this be attributed to toxicity or hormonal inhibition?

Answer:We appreciate the insightful question raised by the reviewer regarding the mechanism behind the reduced efficacy at high CPPU concentrations. We unanimously agree that toxicity and hormone suppression are both reasonable explanations, and we have now expanded our discussion on this point in the revised manuscript to more thoroughly address this issue.

ABA, as a typical stress-responsive hormone, enhances plant adaptation to adversity by inducing stomatal closure. In this study, when the CPPU treatment concentration exceeded 10 mg·L-1, ABA levels increased significantly and continued to accumulate. This accumulation may have restricted photosynthetic carbon assimilation by inhibiting leaf gas exchange, which could be an important reason for the hindered root development in licorice under high CPPU concentrations (20 and 40 mg·L-1). Additionally, high concentrations of CPPU led to a significant reduction in RuBP carboxylase activity, further indicating that elevated CPPU levels may exert toxic effects on licorice plants.

10. In Figure 1, was any correlation or regression analysis conducted between RuBP activity and root growth indices?

Answer: We thank the reviewer for raising this interesting point. we acknowledge that exploring such relationships is an important aspect of mechanistic understanding. Upon careful consideration, we determined that a formal correlation analysis between RuBP activity and root growth indices may not be the most direct approach to address the primary objective of this study for the following reasons:

Focus on Treatment Effects: The main goal of Figure 1 was to demonstrate the direct impact of CPPU concentration gradients on a suite of independent physiological parameters (including RuBP activity), rather than to establish a predictive relationship between these parameters themselves. Multifactorial Regulation: Root growth is a complex trait regulated by multiple interdependent factors (hormone levels). Isolating a direct correlation with one enzymatic activity might oversimplify the mechanism and could be confounded by other variables.

11. The English style needs to be revised along the manuscript.

Answer: We thank the reviewer for this important comment. We agree that the manuscript would benefit from professional English language polishing. In response, the entire manuscript has been thoroughly revised by a native English-speaking colleague with expertise in plant science. We have carefully checked and improved the grammar, vocabulary, sentence flow, and overall academic style to ensure clarity and readability. We believe the revised version now meets the journal's high standards for language quality.

12. Check the references in the style of the journal

Answer: We thank the reviewer for this reminder. We have carefully checked and reformatted all references throughout the manuscript to ensure they strictly adhere to the journal's prescribed style guide

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscript ID: sustainability-3956743

Manuscript Title: Forchlorfenuron as a safe growth regulator significantly improves yield and quality of Glycyrrhiza uralensis

Journal Name: Sustainability

Reviewer Comments

Subject and title

This study is highly significant as it provides a practical and sustainable solution to the decline of natural licorice resources by enabling its high-yield and high-quality cultivation. The thorough assessment of CPPU residues and consumption safety ensures its safe application, establishing a solid foundation for sustainable cultivation that meets growing demand for Glycyrrhiza roots. However, the following comments and suggestions should be considered in the revised manuscript.

Abstract

Line 18-20: Rephrase to: "The most pronounced effects were observed with 10 mg·L⁻¹ CPPU, which increased root yield by 46%, glycyrrhizic acid content by 90%, and liquiritin content by 89%."

Line 20: Correct to: "… it significantly elevated ribulose-1,5-bisphosphate (RuBP) carboxylase activity …".

Line 22-24: The abstract mentions "consumption safety" but only presents the residue level and half-life. The critical result that the "health risk to consumers is negligible" is stated but not quantitatively supported within the abstract itself. Include a key quantitative safety metric in the abstract.

Line 25: The phrase "...posing no health risk to consumers" is a very definitive and absolute statement. In scientific risk assessment, we typically state that the risk is "negligible" or "below levels of concern" rather than absolute zero. Rephrase to a more scientifically precise statement, such as: "...indicating a negligible health risk to consumers." or "...posing no significant health risk to consumers.". Also, add scientific evidence such as (RQ < 1).

Introduction

Line 35: Rephrase to: "Consequently, licorice root is widely recognized and utilized as a functional food [11]."

Line 37-38: Rewrite the sentence to align with the journal scope. For example: "Consequently, the cultivation of G. uralensis has been initiated as a sustainable alternative to wild harvesting [12-13]."

Line 41-43: Rephrase the sentence "In fact, in some cultivated populations..." to strength the connection within the context. I suggest: "In some cases, the content of medicinal ingredients even fails to meet the standards set by the ..."

Line 47: Delete "artificially", rewrite to: "...a class of synthesized exogenous substances..."

Line 49: The transition from the problem (low yield/quality of cultivated licorice) to the proposed solution (CPPU) is somewhat abrupt. The rationale for why a cytokinin-like regulator would be particularly suitable for licorice is not fully established. Add a sentence explaining that enhancing the growth of underground storage organs (like licorice roots) and the synthesis of secondary metabolites could be effectively targeted by regulators influencing cell division and metabolic pathways, such as cytokinins (add recent citations).

Line 52-54: Rephrase to show the specific knowledge gap: "However, current research on CPPU predominantly focuses on its effects on fruit yield, while its impact on medicinal plants, particularly on root biomass and secondary metabolite accumulation, has seldom been systematically investigated."

Line 55-56: Rephrase to: " In recent years, the excessive use of plant growth regulators in agriculture has raised public concern about food safety (add citation)."

Line 60, 61: Add related references.

Line 62-63: Narrow the stated scope here to match the manuscript's content. Replace "long-term safety concerning ecology and health" with "safety through residue dissipation dynamics and dietary risk assessment."

Line 69-72: Rephrase to: "The findings are expected to provide a theoretical foundation for enhancing the cultivation of G. uralensis and to offer practical insights for the safe and effective use of plant growth regulators in medicinal plant agriculture."

Materials and Methods

Line 77: Add the affiliation of prof. Ma Miao.

Line 88: The preparation of the sandy soil substrate is not described. Provide basic information on the growth substrate (e.g., pH, organic matter, nutrient content, EC).

Line 90-92: Rephrase to: "Foliar applications commenced when the seedlings had developed eight true leaves. Each plant was sprayed with 100 mL of either distilled water (control) or a CPPU solution at concentrations of..."

Line 92: The experimental design mentions spraying concentrations of 2.5, 5, 10, 20, and 40 mg·L⁻¹, but the abstract focus on 0, 5, 10, and 20 mg·L⁻¹. This inconsistency is confusing for the reader. Justify the wider range in the methods or maintain consistency by stating the same concentration range (0, 5, 10, 20 mg·L⁻¹) across all sections for clarity.

Line 96: How many plants are in each replicate?

Line 97: Correct to: "ribulose-1,5-bisphosphate (RuBP) carboxylase"

Line 104: What is the age of plants when estimating the relative chlorophyll content and photosynthetic gas exchange parameters?

Line 104: Rephrase to: "For photosynthetic measurements, six plants per treatment were randomly selected."

Line 105-106: Controlling the carbon dioxide concentration and light intensity requires conducting the experiment inside a specially designed greenhouse. Please describe the conditions in the location where the experiment was conducted.

Line 118-119: Rewrite to: "The second fully expanded leaf from the apex of randomly selected seedlings was collected and immediately frozen in liquid nitrogen"

Line 127-128: These full words of UPLC-MS/MS should be moved to line 115 (the first mention in the text).

Line 148-155: Please add a reference to this method (total flavonoid determination).

Line 165: "glycyrrhizin" is the same as "glycyrrhizic acid." Replace "glycyrrhizin" with "liquiritin"

Line 171: Were the roots heated before drying at 60 °C, or is this phrase (were heated) redundant?

Line 178-185: Please add a reference to this method (CPPU determination).

Results

Line 228: Rewrite: "Most photosynthetic parameters exhibited a hormetic response to CPPU concentration, increasing to an optimum at 10 mg·L⁻¹ before declining at higher concentrations; intercellular CO₂ concentration (Ci) was the exception to this trend."

Line 238: Correct to: "RuBP"

Line 252: Rephrase to: "Visual assessment and quantitative measurements confirmed that CPPU treatments significantly influenced the growth architecture of G. uralensis plants (Figure 3)." This provides context for what Figure 3 shows (both a visual and a quantitative representation of growth).

Line 262: Delete the space in "represent significant differences"

Line 265: Correct to: "initially increased before subsequently decreasing"

Line 266: Rephrase to: "The treatment with 10 mg·L⁻¹ CPPU significantly enhanced root biomass to a value 1.46 times greater than that of the control (Table 3)."

Line 270: These values (23.76, 48.51, and 174.11) expressed the yield or productivity not the content. The content/concentration can be expressed as mg/g DW. Please add that the results of the bioactive compounds content here may differ from their productivity/yield results due to the difference in root biomass in response to various CPPU levels.

Line 271: Correct to: "mg/plant, respectively"

Line 272: Correct table number "Table 3"

Line 273: In Table 3, correct to: "Biomass root (g/plant DW)". Also, for all metabolites, add DW to their units (mg/g DW).

Line 278-279: Correct the unit (mg·L⁻¹) and improve this sentence. For example: "By day 56, the CPPU residue in the roots had dissipated to 5.44 × 10⁻⁴ mg·kg⁻¹, representing only 8% of the maximum residue level detected."

Discussion

Line 320: This definition (chlorophenylurea) should be added early in the Introduction section together with forchlorfenuron.

Line 329: "glycyrrhizin" is the same as "glycyrrhizic acid." Replace "glycyrrhizin" with "liquiritin"

Line 331: Rewrite to: "The concurrent increase in root biomass and key bioactive compounds demonstrates that CPPU can not only enhance the yield of fruit crops, but also..."

Line 335-336: Rephrase to: "The application of plant growth regulators is known to alter endogenous hormone homeostasis, thereby influencing plant metabolism and growth (add citations)."

Line 339: This English name (Ural licorice) should be mentioned early in the Introduction beside the scientific name (Glycyrrhiza uralensis).

Line 342-344: Rewrite to improve the sentence: "The inhibition of growth and photosynthesis observed at high CPPU concentrations (20 and 40 mg·L⁻¹) may be attributed to the concomitant sharp increase in leaf abscisic acid, a hormone known to suppress growth and induce stomatal closure."

Line 344: Add a relevant previous study to support increased abscisic acid biosynthesis in response to high concentrations of CPPU.

Line 347: Explain the relationship between increased concentration of endogenous cytokinin (zeatin) and CPPU application (use relevant citation).

Line 352-354: The data shows CPPU increased RuBP carboxylase activity (a dark reaction enzyme) and gas exchange parameters, not necessarily photochemical reactions (like PSII efficiency). Therefore, align the conclusion strictly with your data. For example: "This finding suggests that CPPU enhances the photosynthetic carbon assimilation capacity of G. uralensis by promoting the activity of RuBP carboxylase and stomatal conductance."

Line 363: The comparison of CPPU half-life in licorice roots with fruits (melon, grape) is good, but the explanation for why the half-life is longer in roots is missing. This is a crucial point for understanding residue dynamics in root crops. The authors can suggest that: "The longer dissipation half-life observed in licorice roots (11.74 days) compared to fruits is likely due to the root being a storage organ with lower metabolic activity and different vascular connectivity compared to rapidly developing fruits, leading to slower compound degradation."

Conclusion

While the discussion ends with a summary, a formal conclusion serves distinct and vital purposes. The discussion ends by simply repeating the main results. A strong conclusion should synthesize the findings and highlight their broader significance for sustainable agriculture and medicinal plant cultivation.

References

According to the journal's requirements, journal names should be formatted as abbreviated and italicized.

Author Response

Abstract

1. Line 18-20: Rephrase to: "The most pronounced effects were observed with 10 mg·L⁻¹ CPPU, which increased root yield by 46%, glycyrrhizic acid content by 90%, and liquiritin content by 89%."

Answer: Based on your suggestion, we have rewritten the original sentence

2. Line 20: Correct to: "… it significantly elevated ribulose-1,5-bisphosphate (RuBP) carboxylase activity …".

Thank you for your effort. We have made revisions and corrected the entire text in this regard

3. Line 22-24: The abstract mentions "consumption safety" but only presents the residue level and half-life. The critical result that the "health risk to consumers is negligible" is stated but not quantitatively supported within the abstract itself. Include a key quantitative safety metric

Based on your suggestion, we have increased the RQ valuein the abstract.

4. Line 25: The phrase "...posing no health risk to consumers" is a very definitive and absolute statement. In scientific risk assessment, we typically state that the risk is "negligible" or "below levels of concern" rather than absolute zero. Rephrase to a more scientifically precise statement, such as: "...indicating a negligible health risk to consumers." or "...posing no significant health risk to consumers.". Also, add scientific evidence such as (RQ < 1).

Based on your suggestion, we have made modifications and added RQ values

Introduction

5. Line 35: Rephrase to: "Consequently, licorice root is widely recognized and utilized as a functional food [11]."

We have rewritten it according to your suggestion

6. Line 37-38: Rewrite the sentence to align with the journal scope. For example: "Consequently, the cultivation of  uralensishas been initiated as a sustainable alternative to wild harvesting [12-13]."

We have rewritten it according to your suggestion

7. Line 41-43: Rephrase the sentence "In fact, in some cultivated populations..." to strength the connection within the context. I suggest: "In some cases, the content of medicinal ingredients even fails to meet the standards set by the ..."

We have rewritten it according to your suggestion

8. Line 47: Delete "artificially", rewrite to: "...a class of synthesized exogenous substances..."

Based on your suggestion, we have removed the word

9. Line 49: The transition from the problem (low yield/quality of cultivated licorice) to the proposed solution (CPPU) is somewhat abrupt. The rationale for why a cytokinin-like regulator would be particularly suitable for licorice is not fully established. Add a sentence explaining that enhancing the growth of underground storage organs (like licorice roots) and the synthesis of secondary metabolites could be effectively targeted by regulators influencing cell division and metabolic pathways, such as cytokinins (add recent citations).

We added a sentence to transition from low yield/quality cultivation of licorice to proposed solutions

10. Line 52-54: Rephrase to show the specific knowledge gap: "However, current research on CPPU predominantly focuses on its effects on fruit yield, while its impact on medicinal plants, particularly on root biomass and secondary metabolite accumulation, has seldom been systematically investigated."

Based on your suggestion, we have rephrased the sentence

11. Line 55-56: Rephrase to: " In recent years, the excessive use of plant growth regulators in agriculture has raised public concern about food safety (add citation)."

Based on your suggestion, we have rephrased this section and added citations

12. Line 60, 61: Add related references.

Based on your suggestion, we have added citations

13. Line 62-63: Narrow the stated scope here to match the manuscript's content. Replace "long-term safety concerning ecology and health" with "safety through residue dissipation

Based on your suggestion, we have completed the replacementdynamics and dietary risk assessment."

14. Line 69-72: Rephrase to: "The findings are expected to provide a theoretical foundation for enhancing the cultivation of  uralensisand to offer practical insights for the safe and effective use of plant growth regulators in medicinal plant agriculture."

Based on your suggestion, we have rewritten this paragraph

Materials and Methods

15. Line 77: Add the affiliation of prof. Ma Miao.

We have added Professor Ma Miao's affiliation.

16. Line 88: The preparation of the sandy soil substrate is not described. Provide basic information on the growth substrate (e.g., pH, organic matter, nutrient content, EC).

We provide basic information about the growth substrate in the methods section, such as pH value, organic matter, and nutrient composition.

17. Line 90-92: Rephrase to: "Foliar applications commenced when the seedlings had developed eight true leaves. Each plant was sprayed with 100 mL of either distilled water (control) or a CPPU solution at concentrations of..."

According to your request, we have rewritten this part of the content

18. Line 92: The experimental design mentions spraying concentrations of 2.5, 5, 10, 20, and 40 mg·L⁻¹, but the abstract focus on 0, 5, 10, and 20 mg·L⁻¹. This inconsistency is confusing for the reader. Justify the wider range in the methods or maintain consistency by stating the same concentration range (0, 5, 10, 20 mg·L⁻¹) across all sections for clarity.

We have corrected the content in the abstract and checked the entire text

19. Line 96: How many plants are in each replicate?

4 pots per treatment, 4 seedlings per pot, a total of 16 seedlings per treatment

20. Line 97: Correct to: "ribulose-1,5-bisphosphate (RuBP) carboxylase"

Based on your suggestion, we have corrected the name and checked the entire text

21. Line 104: What is the age of plants when estimating the relative chlorophyll content and photosynthetic gas exchange parameters?

Answer: It takes about a month for four true leaves, and about 50 days to reach the stage of eight true leaves. We processed it for a week and measured the photosynthetic gas exchange parameters after one week. So the age of the plants at this time is about 70 days

22. Line 104: Rephrase to: "For photosynthetic measurements, six plants per treatment were randomly selected."

Based on your suggestion, we have rewritten the content here

23. Line 105-106: Controlling the carbon dioxide concentration and light intensity requires conducting the experiment inside a specially designed greenhouse. Please describe the conditions in the location where the experiment was conducted.

Answer: We measured the photosynthetic gas exchange parameters in an outdoor environment. The LI-6400 photosynthetic apparatus we use comes with an artificial light source and a CO₂ cylinder. We have added descriptions in the text to avoid ambiguity

24. Line 118-119: Rewrite to: "The second fully expanded leaf from the apex of randomly selected seedlings was collected and immediately frozen in liquid nitrogen"

Based on your suggestion, we have rewritten this part of the content

25. Line 127-128: These full words of UPLC-MS/MS should be moved to line 115 (the first mention in the text).

Answer: We will move these complete words of UPLC-MS/MS to their first appearance

26. Line 148-155: Please add a reference to this method (total flavonoid determination).

Based on your feedback, we have added references here

27. Line 165: "glycyrrhizin" is the same as "glycyrrhizic acid." Replace "glycyrrhizin" with "liquiritin"

Thank you for pointing out this error. We have made a decision

28. Line 171: Were the roots heated before drying at 60 °C, or is this phrase (were heated) redundant?

Thank you for your suggestion. We have removed the redundant parts

29. Line 178-185: Please add a reference to this method (CPPU determination).

We have added references here

 

Results

30. Line 228: Rewrite: "Most photosynthetic parameters exhibited a hormetic response to CPPU concentration, increasing to an optimum at 10 mg·L⁻¹ before declining at higher concentrations; intercellular CO₂ concentration (Ci) was the exception to this trend."

Based on your suggestion, we have made revisions to this section

31. Line 238: Correct to: "RuBP"

We have corrected this part of the content

32. Line 252: Rephrase to: "Visual assessment and quantitative measurements confirmed that CPPU treatments significantly influenced the growth architecture of  uralensisplants (Figure 3)." This provides context for what Figure 3 shows (both a visual and a quantitative representation of growth).

Based on your suggestion, we have rewritten the content here

33. Line 262: Delete the space in "represent significant differences"

We have removed the spaces in the table

34. Line 265: Correct to: "initially increased before subsequently decreasing"

Based on your suggestion, we have corrected this part of the content

35. Line 266: Rephrase to: "The treatment with 10 mg·L⁻¹ CPPU significantly enhanced root biomass to a value 1.46 times greater than that of the control (Table 3)."

Based on your suggestion, we have rewritten the content here

36. Line 270: These values (23.76, 48.51, and 174.11) expressed the yield or productivity not the content. The content/concentration can be expressed as mg/g DW. Please add that the results of the bioactive compounds content here may differ from their productivity/yield results due toDear reviewer, we have removed the section on yield or productivity.

Considering our main idea, we will present the content on yield in future field experiments the difference in root biomass in response to various CPPU levels.

37. Line 271: Correct to: "mg/plant, respectively"

Considering our main idea, we will present the content on yield in future field experiments the difference in root biomass in response to various CPPU levels.

38. Line 272: Correct table number "Table 3"

Based on your suggestion, we have corrected this part of the content

39. Line 273: In Table 3, correct to: "Biomass root (g/plant DW)". Also, for all metabolites, add DW to their units (mg/g DW).

Considering our main idea, we will present the content on yield in future field experiments the difference in root biomass in response to various CPPU levels.

 

40. Line 278-279: Correct the unit (mg·L⁻¹) and improve this sentence. For example: "By day 56, the CPPU residue in the roots had dissipated to 5.44 × 10⁻⁴ mg·kg⁻¹, representing only 8% of the maximum residue level detected."

Based on your suggestion, we have corrected this part of the content

Discussion

41. Line 320: This definition (chlorophenylurea) should be added early in the Introduction section together with forchlorfenuron.

Answer: We have placed the definition of chloroquine in the introduction section.

42. Line 329: "glycyrrhizin" is the same as "glycyrrhizic acid." Replace "glycyrrhizin" with "liquiritin"

Based on your suggestion, we have corrected this part of the content

43. Line 331: Rewrite to: "The concurrent increase in root biomass and key bioactive compounds demonstrates that CPPU can not only enhance the yield of fruit crops, but also..."

Based on your suggestion, we have rewritten the content here

 

44. Line 335-336: Rephrase to: "The application of plant growth regulators is known to alter endogenous hormone homeostasis, thereby influencing plant metabolism and growth (add citations)."

We have cited references in this section

45. Line 339: This English name (Ural licorice) should be mentioned early in the Introduction beside the scientific name (Glycyrrhiza uralensis).

Thank you for your reminder. Ural licorice has been removed and the entire text has been checked

46. Line 342-344: Rewrite to improve the sentence: "The inhibition of growth and photosynthesis observed at high CPPU concentrations (20 and 40 mg·L⁻¹) may be attributed to the concomitant sharp increase in leaf abscisic acid, a hormone known to suppress growth and induce stomatal closure."

We have rewritten this section based on your suggestion

47. Line 344: Add a relevant previous study to support increased abscisic acid biosynthesis in response to high concentrations of CPPU.

Answer: We did not find any literature related to the impact of CPPU on ABA

48. Line 347: Explain the relationship between increased concentration of endogenous cytokinin (zeatin) and CPPU application (use relevant citation).

Answer: We did not find any literature related to the impact of CPPU on ABA

49. Line 352-354: The data shows CPPU increased RuBP carboxylase activity (a dark reaction enzyme) and gas exchange parameters, not necessarily photochemical reactions (like PSII efficiency). Therefore, align the conclusion strictly with your data. For example: "This finding suggests that CPPU enhances the photosynthetic carbon assimilation capacity of  uralensisby promoting the activity of RuBP carboxylase and stomatal conductance."

Thank you for your reminder. We have revised this section based on your suggestion

50. Line 363: The comparison of CPPU half-life in licorice roots with fruits (melon, grape) is good, but the explanation for why the half-life is longer in roots is missing. This is a crucial point for understanding residue dynamics in root crops. The authors can suggest that: "The longer dissipation half-life observed in licorice roots (11.74 days) compared to fruits is likely due to the root being a storage organ with lower metabolic activity and different vascular connectivity compared to rapidly developing fruits, leading to slower compound degradation."

Thank you for your suggestion. We have added this section in the corresponding location

Conclusion

51. While the discussion ends with a summary, a formal conclusion serves distinct and vital purposes. The discussion ends by simply repeating the main results. A strong conclusion should synthesize the findings and highlight their broader significance for sustainable agriculture and medicinal plant cultivation.

We have revised the conclusion to highlight its significance in sustainable agriculture and medicinal plant cultivation

References

52. According to the journal's requirements, journal names should be formatted as abbreviated and italicized.

We have rechecked and revised the references in accordance with the requirements of the journal

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors must undertake a comprehensive re-evaluation of this work, beginning with a full audit and verification of all primary data—particularly the catastrophic discrepancy in CPPU residue values—and, if necessary, repeat key experiments. The study must be substantially strengthened by rigorously expanding the Methods for reproducibility, reforming the statistical analysis with complete reporting standards, and providing a mechanistic discussion that moves beyond descriptive results to propose testable hypotheses.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English of the manuscript is understandable but requires significant improvement to meet the standards of an international academic journal. Numerous instances of non-idiomatic phrasing, grammatical errors, and awkward sentence construction hinder readability and professional presentation.

I strongly recommend that the manuscript undergo thorough editing by a professional language editing service or a native English speaker with expertise in plant sciences. This will greatly enhance the clarity, fluency, and overall quality of the presentation.

Author Response

We extend our sincere gratitude to the reviewer for the considerable time and effort invested in reviewing our manuscript. Your insightful comments and constructive suggestions have been invaluable in helping us to significantly improve the quality of our work.

1. Critical Data Inconsistency and Lack of Integrity:

The most severe issue is the eight-order-of-magnitude discrepancy in the reported residue level of CPPU, a key finding. The Abstract states a residue of 5.44×10⁴ mg/kg , while the Results section (Figure 5, Section 2.5, Table, Table 5) consistently reports 5.44×10^-4 mg/kg. This is not a minor typographical error but a catastrophic failure in data presentation and verification. The safety conclusion of "negligible health risk" is entirely dependent on which of these values is correct. The authors' annotation that the value "has been corrected" is insufficient; without a clear explanation for the source of this error and evidence of a full, top-to-bottom audit of the entire dataset, the credibility of the entire study cannot be trusted. This single issue is grounds for immediate rejection.

We thank the reviewer for raising this point, which highlights an inconsistency in presentation rather than a factual error in the data. In the abstract, we presented the value as approximately 5 × 10⁻⁴ mg/kg, which is a legitimate simplification of the more precise value 5.44 × 10⁻⁴ mg/kg used in the Results section. Both expressions refer to the same order of magnitude (10⁻⁴). However, we fully acknowledge that using different formats created unnecessary confusion and could be misinterpreted as a drastic discrepancy. To eliminate any possible ambiguity and ensure absolute consistency, we have now revised the abstract to use the precise value of 5.44 × 10⁻⁴ mg/kg, making it identical to the value reported in the Results and figures.

2. Inadequate Statistical Reporting and Experimental Design:

The statistical analysis lacks the necessary detail for critical evaluation. Reporting only P-values and using letters on figures without providing full ANOVA summaries (F-values, degrees of freedom) is substandard for a scientific publication. Furthermore, the description of biological replicates is ambiguous (e.g., "eight plants were randomly selected"), blurring the distinction between true independent replicates independent replicates and sub-samples. This ambiguity calls into question the robustness and reliability of the statistical inferences drawn.

 

Response: We thank the reviewer for this constructive feedback. We have comprehensively addressed both points in the revised manuscript.

 

1. Regarding statistical reporting:

As recommended, we have supplemented the full ANOVA summaries—including F-values and degrees of freedom—for all key analyses in the Results section. For example, concerning photosynthetic parameters, the ANOVA results demonstrate that the CPPU treatments had highly significant effects on: photosynthetic rate (Pn; F(5,30) = 7.785, P < 0.001), transpiration rate (Tr; F(5,30) = 9.646, P < 0.001), stomatal conductance (Gs; F(5,30) = 9.694, P < 0.001), and ribulose-1,5-bisphosphate carboxylase enzyme activity (RuBPCase; F(5,30) = 53.899, P < 0.001). Providing these complete statistical details enhances the rigor and evaluability of our findings.

 

2. Clarification on Biological Replicates and Sampling:

We appreciate the reviewer’s attention to the description of biological replicates. To eliminate ambiguity, we now explicitly define the pot as the independent biological replicate (n = 4 per treatment). To ensure accurate representation of variation at the appropriate experimental-unit level, we adopted a nested sampling approach: two plants were randomly selected from each of the four replicate pots per treatment, yielding a total of eight plants measured per treatment. This clarifies that all statistical inferences are correctly based on the pot as the experimental unit, thereby avoiding pseudoreplication. The relevant Methods and Results sections have been updated accordingly.

3. Lack of Mechanistic Depth:

The study remains predominantly descriptive, cataloguing changes in photosynthesis, hormones, and metabolites without providing a deeper, mechanistic explanation. Claims that CPPU "promoted the synthesis" of hormones are speculative and correlative. The Discussion fails to integrate the findings into a coherent physiological model that explains how CPPU orchestrates these multifaceted changes. A more profound interpretation, linking hormonal shifts to gene regulation, metabolic flux, and resource partitioning, is required to elevate the manuscript from an observational report to a substantive scientific contribution.

Response:We fully concur with the reviewer's insights. Accordingly, we have thoroughly restructured the discussion section, moving beyond a mere enumeration of phenomena to propose a ‘model wherein CPPU modulates growth and metabolism via hormonal regulation’, aiming to elucidate how CPPU orchestrates these multidimensional changes. Detailed elaboration on this substantial revision will follow below.

Collectively, our data indicate that CPPU’s function extends beyond simply stimulating growth. It appears to initiate a comprehensive readjustment of the hormonal network within the plant, which in turn guides the direction of metabolic flows. For instance, the marked increase in endogenous zeatin directly reflects the plant’s recognition of the exogenous CPPU signal. Elevated zeatin levels promote cell division and expansion, leading to increased biomass in roots [47]. At the same time, the notable rise in salicylic acid is no coincidence—it functions like a dedicated “metabolic switch”. Recognized as a crucial regulatory factor, salicylic acid activates key enzymes in the phenylpropanoid pathway, thus providing a biologically sound, hormone-based rationale for the concurrent enhancement of glycyrrhizin and flavonoids. We propose that CPPU-triggered salicylic acid serves as a central signaling molecule, transmitting the CPPU stimulus precisely into those biosynthetic routes that improve crop quality. Moreover, the growth inhibition and reduced photosynthesis seen under high CPPU concentrations correspond closely with a stress response provoked by the abrupt surge in ABA, which is known to cause stomatal closure [48]. Taken together, these interconnected hormonal adjustments constitute an integrated regulatory framework through which CPPU skillfully harmonizes both structural growth and the accumulation of specialized metabolites. Additionally, high concentrations of CPPU led to a significant reduction in RuBP carboxylase activity, further indicating that elevated CPPU levels may exert toxic effects on licorice plants.

4. Insufficient Methodological Detail for Reproducibility:

Key details required for the replication of the experiments are missing. The description of the growth substrate ("sandy soil") is inadequate without basic characterization (pH, organic matter, nutrient content). Essential parameters for analytical methods, such as recovery rates and limits of detection/quantification (LOD/LOQ) for the UPLC-MS/MS analyses, are omitted, preventing a full assessment of the data's reliability.

Response:We thank the reviewer for this valuable comment. The reviewer rightly pointed out that the characterization of the growth substrate is crucial for experimental replication. We apologize for any confusion caused by the initial description.

In our original manuscript, the detailed physicochemical properties of the soil mixture used in the main pot experiment were provided in the same Methods section, following the description of the potting procedure. These seedlings were transplanted together into a single plastic pot (23 cm height × 20 cm diameter) containing a sterilized substrate mixture of river sand and loam (3:7, v/v). The substrate had the following properties: pH 7.8; total nitrogen, phosphorus, and potassium concentrations were 0.315, 0.131 and 5.47 g·kg−1, respectively; available nitrogen, phosphorus, and potassium levels were 52.59, 5.23 and 50.04 mg·kg−1, respectively; and the organic matter content was 6.64 g·kg−1. 

Response:Thank you very much for your valuable comments and attention to the reliability of the research findings.

The issue you raised regarding the methodological parameters of UPLC-MS/MS analysis (such as recovery rates, LOD/LOQ) is of great importance. We would like to clarify that the core objective of this study is to investigate the effects of CPPU treatment on plant growth and quality, rather than to develop a new analytical method. Therefore, the UPLC-MS/MS detection method employed in this study fully follows a well-established and published protocol. In the cited original literature, the authors have systematically and rigorously validated a complete set of methodological parameters, including recovery rates, LOD, LOQ, linear range, and precision. This method has been widely recognized and applied in the field. We believe that directly referencing such a thoroughly validated and mature method can maximize the accuracy and comparability of our experimental data. Additionally, we have indicated the relevant parameters of the method, such as recovery rates and LOD/LOQ, in the main text.

Thank you again for your review, and we hope the above explanation addresses your concerns.

5. Presentation and Language:

The manuscript requires extensive language polishing to meet the standards of an international scientific journal. Figures are of low quality, and table layouts are confusing, hindering comprehension.

We sincerely thank the reviewer for their valuable feedback. We have thoroughly addressed all the points raised:

Language Polishing: The entire manuscript has undergone professional English editing to meet international standards.

Figure Quality: All figures have been replaced with new, high-resolution versions for optimal clarity.

Table Layout: All tables have been redesigned to be clear and reader-friendly.

We believe these revisions have significantly improved the manuscript. All changes have been highlighted in the text for your convenience. Thank you again for your helpful comments.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have made substantial improvements to the manuscript, effectively addressing the most critical concerns from the previous review, particularly the data integrity of the CPPU residue values. The study now presents a robust and valuable contribution. To elevate the manuscript to its final publishable standard, we recommend:

1. Deepen the Discussion: Further refine the mechanistic interpretation by more clearly distinguishing your direct findings from the well-informed, literature-based hypotheses you propose for the underlying physiology.

2. Professional Language Edit: A final, thorough polish of the English language by a native speaker or professional service is essential for clarity and fluency.

3. Refine Figure 3: Ensure high-resolution images and correct the labels to include a space between the numerical value and the unit (e.g., "10 mg L⁻¹").

We are confident that addressing these points will finalize a very strong manuscript for publication.

Comments on the Quality of English Language

The English of the manuscript is understandable but requires significant improvement to meet the standards of an international academic journal. Numerous instances of non-idiomatic phrasing, grammatical errors, and awkward sentence construction hinder readability and professional presentation.

I strongly recommend that the manuscript undergo thorough editing by a professional language editing service or a native English speaker with expertise in plant sciences. This will greatly enhance the clarity, fluency, and overall quality of the presentation.

Author Response

We extend our sincere gratitude to the reviewer for the considerable time and effort invested in reviewing our manuscript. Your insightful comments and constructive suggestions have been invaluable in helping us to significantly improve the quality of our work.

 

1. Deepen the Discussion: Further refine the mechanistic interpretation by more clearly distinguishing your direct findings from the well-informed, literature-based hypotheses you propose for the underlying physiology.

Response: Based on your suggestion, we will directly distinguish it from potential physiological hypotheses and further improve the mechanism explanation. Below is the revised content for your reference

Our findings indicate that CPPU application induces a series of interconnected physiological responses in licorice. At the optimal concentration of 10 mg·L-1, leaf photosynthetic capacity was substantially enhanced, accompanied by a marked increase in RuBPCase activity to 161.29% of the control level. Unlike its documented role in enhancing chlorophyll content in Dioscorea opposita Thunb. [45] or delaying chlorophyll degradation in banana [46], the positive effect of CPPU on the photosynthetic system of licorice appears to be mediated principally through the upregulation of RuBPCase. The synchronized increase in both parameters suggests that RuBPCase activation plays a central mediating role in the CPPU induced stimulation of photosynthetic carbon assimilation in this species.

The role of CPPU extends far beyond merely promoting growth; it recalibrates the hormonal network within the plant, thereby directing the flow of metabolic processes [47]. This study directly documents three core observations: (1) a significant increase in endogenous zeatin levels; (2) a sharp a sharp rise in salicylic acid (SA) content; and (3) under high-CPPU treatment, a substantial accumulation of abscisic acid (ABA) accompanied by severe suppression of photosynthesis. First, the marked elevation in endogenous zeatin provides direct evidence that the plant perceives the external CPPU signal. Given that zeatin is well established to stimulate cell division and expansion [48], we propose that its upregulated levels offer a plausible hormonal explanation for the observed increase in root biomass. Second, the rapid increase in SA coincided with the accumulation of glycyrrhizin and flavonoids. Based on the consensus that SA is a key regulator of the phenylpropanoid pathway [49], we hypothesize that CPPU-induced SA acts as a central signaling molecule. Functioning as a precise "metabolic switch," it activates key enzymes in this pathway, transducing the upstream signal into downstream biosynthetic routes that enhance crop quality. Moreover, the growth inhibition and photosynthetic decline induced by high CPPU levels strongly correlated with the explosive increase in ABA. Extensive studies have demonstrated that ABA induces stomatal closure, thereby impairing photosynthesis [50]. We therefore conclude that high CPPU concentrations likely trigger excessive ABA synthesis, forcing the plant into a stressed state that ultimately restrains growth. In summary, these interrelated hormonal shifts collectively form an integrated regulatory framework. Through this framework, CPPU artfully coordinates structural growth with the accumulation of specialized metabolites. It should also be emphasized that high CPPU levels significantly reduced RuBPCase activity, implying that supraoptimal CPPU may exert phytotoxic effects on licorice plants.

2. Professional Language Edit: A final, thorough polish of the English language by a native speaker or professional service is essential for clarity and fluency.

Response: Our English has been thoroughly polished by professional service personnel to ensure clarity and fluency.

 

3. Refine Figure 3: Ensure high-resolution images and correct the labels to include a space between the numerical value and the unit (e.g., "10 mg L⁻¹").

Reply: We ensure that the image is a high-resolution image and have corrected the image label to include spaces between values and units

Author Response File: Author Response.pdf