Cytokinin Oxidase/Dehydrogenase 1 (FvCKX1) Coordinates Receptacle Growth and Achene Maturation in Strawberry

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Cytokinin oxidase/dehydrogenase 1 (FvCKX1) coordinates the receptacle growth and the achene maturation in Fragaria vesca”, authored by Yunhe Tian investigates the molecular mechanisms coordinating seed (achene) maturation and receptacle growth during strawberry fruit development. Using RNA-seq analysis during the ripening transition phase, the study identifies differentially expressed genes between achenes and receptacles and highlights FvCKX1, a cytokinin degradation gene, as a key regulatory factor. Functional validation through transient RNA interference and overexpression experiments demonstrates that suppression of FvCKX1 promotes receptacle enlargement but delays achene maturation, whereas overexpression has the opposite effect, indicating a regulatory role in balancing fruit tissue development. The most interesting finding is that FvCKX1 appears to coordinate fruit tissue development partly by negatively regulating abscisic acid (ABA) accumulation in the receptacle, thereby linking cytokinin metabolism with hormonal control of strawberry fruit ripening and growth.

The manuscript addresses an important question in plant developmental biology and horticultural science: the coordination between seed development and fruit tissue growth in non-climacteric fruits such as strawberry. By identifying FvCKX1 as a regulatory component linking cytokinin metabolism with ABA-mediated fruit development, the study contributes to the broader understanding of hormonal interactions controlling fruit growth and ripening. If confirmed by additional studies, these results could influence research in fruit crop improvement, particularly in optimizing fruit size, ripening timing, and seed development. 

The conclusions are generally consistent with the experimental data and align with the objectives outlined at the beginning of the manuscript. The authors successfully demonstrate that FvCKX1 influences receptacle growth and achene maturation, and they propose a model involving hormonal interactions between cytokinin metabolism and ABA accumulation. While the conclusions are reasonable, they could be strengthened by discussing potential alternative explanations and by acknowledging the limitations associated with transient transformation experiments.

To help the authors in the second version, I drew some queries to be anwered. These questions must be answered in the letter but mainly inside the manuscript.
Q1. Can the authors clarify the mechanistic link between FvCKX1 activity and ABA accumulation in the receptacle?
Q2. Why did the authors observe minimal changes in zeatin levels despite manipulating a cytokinin degradation gene?
Q3. Would stable transgenic lines confirm the phenotypic effects observed in transient RNAi and overexpression assays?
Q4. Can the authors provide additional statistical information (exact p-values, biological replicates, and experimental repeats) for the phenotypic analyses?
Q5. Could environmental factors or developmental timing influence the observed hormonal responses?
Q6. Could the authors further explain the biological significance of the antagonistic gene expression patterns observed between achenes and receptacles?
Q7. Were other members of the CKX gene family tested functionally to determine whether they also influence fruit development?
Q8. Can the authors elaborate on the potential interactions between cytokinin, auxin, and ABA signaling pathways during strawberry fruit development?
Q9. What are the potential implications of manipulating FvCKX1 for strawberry breeding or agricultural production?

Overall, this manuscript presents an interesting investigation into the coordination of achene maturation and receptacle growth in strawberry. The integration of transcriptomic data with functional validation experiments provides useful insights into the role of FvCKX1 in fruit development. The study contributes to the understanding of hormonal regulation and tissue coordination during fruit ripening in non-climacteric fruits. However, some methodological clarifications, improved data interpretation, and minor language revisions would strengthen the manuscript and improve its clarity and scientific impact. All questions must be answered inside manuscript.

Author Response

Response to Reviewer 1 Comments

Thanks for the reviewer’s valuable comments and suggestions. Please find the detailed responses below and the corresponding revisions highlighted in track changes in the re-submitted files.

The manuscript entitled “Cytokinin oxidase/dehydrogenase 1 (FvCKX1) coordinates the receptacle growth and the achene maturation in Fragaria vesca”, authored by Yunhe Tian investigates the molecular mechanisms coordinating seed (achene) maturation and receptacle growth during strawberry fruit development. Using RNA-seq analysis during the ripening transition phase, the study identifies differentially expressed genes between achenes and receptacles and highlights FvCKX1, a cytokinin degradation gene, as a key regulatory factor. Functional validation through transient RNA interference and overexpression experiments demonstrates that suppression of FvCKX1 promotes receptacle enlargement but delays achene maturation, whereas overexpression has the opposite effect, indicating a regulatory role in balancing fruit tissue development. The most interesting finding is that FvCKX1 appears to coordinate fruit tissue development partly by negatively regulating abscisic acid (ABA) accumulation in the receptacle, thereby linking cytokinin metabolism with hormonal control of strawberry fruit ripening and growth.

The manuscript addresses an important question in plant developmental biology and horticultural science: the coordination between seed development and fruit tissue growth in non-climacteric fruits such as strawberry. By identifying FvCKX1 as a regulatory component linking cytokinin metabolism with ABA-mediated fruit development, the study contributes to the broader understanding of hormonal interactions controlling fruit growth and ripening. If confirmed by additional studies, these results could influence research in fruit crop improvement, particularly in optimizing fruit size, ripening timing, and seed development. 

The conclusions are generally consistent with the experimental data and align with the objectives outlined at the beginning of the manuscript. The authors successfully demonstrate that FvCKX1 influences receptacle growth and achene maturation, and they propose a model involving hormonal interactions between cytokinin metabolism and ABA accumulation. While the conclusions are reasonable, they could be strengthened by discussing potential alternative explanations and by acknowledging the limitations associated with transient transformation experiments.

To help the authors in the second version, I drew some queries to be answered. These questions must be answered in the letter but mainly inside the manuscript.

Q1. Can the authors clarify the mechanistic link between FvCKX1 activity and ABA accumulation in the receptacle?

Response :We appreciate the reviewer’s insightful question regarding the mechanistic link between FvCKX1 activity and ABA accumulation, which is crucial for understanding how cytokinins (CKs) and ABA collaboratively regulate receptacle development and ripening. Currently, we only observed significant changes in ABA accumulation upon transient manipulation of FvCKX1 expression levels. However, alterations in FvCKX1 expression do not necessarily equate to changes in its enzymatic activity. Thus, whether FvCKX1 activity directly influences ABA accumulation requires further experimental validation. To our knowledge, no studies have reported direct regulation of ABA biosynthesis by FvCKX1. Existing literature consistently indicates that FvCKX1 functions solely through modulating CK homeostasis, and CKs are well-documented antagonists of ABA in multiple biological processes. Therefore, we think the observed changes in CYP707A4a expression and ABA levels likely result from FvCKX1-mediated perturbations in CK content. We have addressed this point in the Discussion section to clarify the indirect nature of this interaction (Lines 426-455).

 

Q2. Why did the authors observe minimal changes in zeatin levels despite manipulating a cytokinin degradation gene?

Response : We appreciate the reviewer’s comment. Here, we transiently knocked down and overexpressed the cytokinin degradation gene FvCKX1 and found no obvious change in trans zeatin content. We speculate that there are two possible reasons for this result. First, other CKX genes in strawberry may be functionally redundant with FvCKX1, and simultaneous knockout of two or more CKX genes may be required to significantly alter zeatin content. Second, plants contain other active cytokinin forms, such as iP. Further detection of other cytokinin forms in strawberry fruits with FvCKX1 overexpression or knockdown will be necessary to confirm whether FvCKX1 regulates strawberry fruit development and ripening by modifying cytokinin levels. We have addressed this point in the Discussion section (Lines 413-425).

Q3. Would stable transgenic lines confirm the phenotypic effects observed in transient RNAi and overexpression assays?

Response : Yes, we fully agree with the reviewer’s suggestion. We will generate stable transgenic lines in future work to confirm the corresponding phenotypic effects.

Q4. Can the authors provide additional statistical information (exact p-values, biological replicates, and experimental repeats) for the phenotypic analyses?

Response : Thanks for the reviewer’s suggestion. We have provided the relevant statistical information at the corresponding locations in the manuscript.

Q5. Could environmental factors or developmental timing influence the observed hormonal responses?

Response : Yes, we think both environmental factors and developmental timing could influence the observed CK and ABA responses. Under natural or experimental conditions, environmental cues such as light intensity, photoperiod, temperature, water status, and nutrient availability are known to modulate the biosynthesis, transport, and signal transduction of CK and ABA. These factors can alter hormone accumulation patterns and downstream physiological outputs independently or interactively. Meanwhile, developmental stage and tissue maturity also play critical roles: hormone sensitivity, metabolic turnover, and regulatory networks often shift dynamically during growth and differentiation, leading to stage-dependent changes in hormonal responses. Accordingly, when investigating hormonal responses in strawberry, all experiments were performed in a greenhouse with fixed environmental conditions, and strawberry fruits at the same developmental stage were used for treatment.

Q6. Could the authors further explain the biological significance of the antagonistic gene expression patterns observed between achenes and receptacles?

Response : Thanks for the reviewer’s insightful question. Strawberry seed dispersal mainly relies on consumption by birds and other animals. Before being consumed, strawberry seeds must first reach maturity, followed by the rapid enlargement and ripening of the receptacle, which attracts animals through its aroma and palatability. Therefore, the coordinated development of strawberry achenes and receptacle is critical for the reproductive success of strawberry progeny. During the strawberry fruit ripening transition, achenes cease growth and undergo maturation. At this stage, achenes must transmit a coordinated signal to the receptacle to trigger rapid expansion and ripening, thereby attracting animals for seed dispersal. Our transcriptome analysis revealed a substantial subset of genes exhibiting opposite expression patterns in achenes versus receptacles during the ripening transition. Given the distinct developmental programs of these two tissues at this stage, we think these genes may mediate coordinated development of achenes and receptacles.

Q7. Were other members of the CKX gene family tested functionally to determine whether they also influence fruit development?

Response: We did not test other FvCKX family members. Since transient overexpression and knockdown of FvCKX1 showed no significant effect on trans-zeatin content, suggesting possible functional redundancy among FvCKX members. Future work should include functional characterization of other FvCKXs and generation of stable overexpression lines and multiplex knockout mutants to elucidate the regulatory roles of FvCKXs in fruit development.

Q8. Can the authors elaborate on the potential interactions between cytokinin, auxin, and ABA signaling pathways during strawberry fruit development?

Response : We appreciate the reviewer’s insightful question. Auxin and gibberellins coordinate fruit development and ripening in strawberry through transcriptional regulation of the ABA metabolic gene CYP707A4a. Auxin and GA from achene in early stages promote the expression of the FveCYP707A4a gene to ensure the endogenous ABA level is extremely low. At the initiation of ripening stage, auxin and GA levels decrease to a threshold, and so does the FveCYP707A4a expression, leading to the activation of the feedforward loop for the steep increase of ABA level to trigger transition from “growing” to “ripening.” The high level of ABA in the late stages further inhibits the expression of FveCYP707A4a to enhance the activation of the feedforward loop (Liao et al, 2018). Intriguingly, the cytokinin catabolic enzyme FvCKX1 emerges as a novel modulator of this process. The expression of FvCKX1 positively correlates with FvCYP707A4a, and genetic manipulation of FvCKX1 reciprocally alters both FvCYP707A4a expression and ABA levels, suggesting cytokinin signaling may fine-tune ABA homeostasis during ripening transition. Since FvCKX1 is a cytokinin (CK) catabolic enzyme, all reported functions of FvCKX1 are achieved through modulating CK homeostasis (Chen et al, 2020; Dob et al, 2021; Zhao et al, 2024; Yang et al, 2025). Therefore, the observed changes in CYP707A4a expression and ABA levels are likely attributed to altered CK levels, given the well-documented antagonistic relationship between CK and ABA in multiple biological processes (Paul E Verslues, 2016; Huang et al, 2018).

Q9. What are the potential implications of manipulating FvCKX1 for strawberry breeding or agricultural production?

Response : CKX genes have been well-documented to regulate seed development (Bartrina et al, 2011; Ashikari et al, 2005; Böttcher et al, 2015). Loss-of-function mutations in Arabidopsis AtCKX3/5 or OsCKX2 in rice or GmCKX3/7/14 in soybean consistently increase seed yield (Bartrina et al, 2011; Ashikari et al, 2005). The manipulation of CKXs holds significant promise for biotechnological applications, with broad implications for agriculture, horticulture, and agroforestry (Gupta et al, 2021). Therefore, FvCKX1 shows strong potential for enhancing strawberry fruit yield and precisely modulating ripening through targeted expression control. Its ABA-mediated effects may additionally enhance stress resilience and fruit quality.

Overall, this manuscript presents an interesting investigation into the coordination of achene maturation and receptacle growth in strawberry. The integration of transcriptomic data with functional validation experiments provides useful insights into the role of FvCKX1 in fruit development. The study contributes to the understanding of hormonal regulation and tissue coordination during fruit ripening in non-climacteric fruits. However, some methodological clarifications, improved data interpretation, and minor language revisions would strengthen the manuscript and improve its clarity and scientific impact. All questions must be answered inside manuscript.

Response : We sincerely appreciate the reviewers' positive evaluation of our manuscript and constructive comments, which have significantly improved the quality of our manuscript and provided valuable guidance for future work. In response to the reviewers' concerns, we have carefully addressed each point and incorporated all suggested revisions in the revised manuscript.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Tian et al. performed RNA-seq analysis on receptacle and achene tissues of Fragaria vesca and identified a key gene, FvCKX1, whose function was preliminarily characterized through RNAi and overexpression experiments. The manuscript is logically structured and presents relatively detailed data. However, several issues require further revision before acceptance:

 

Major points:

1) In the Introduction, the authors discuss the roles of ethylene, auxin, and abscisic acid in fruit development, but cytokinin is not mentioned. Given that this study focuses on CKX, a cytokinin metabolic enzyme, it is recommended that relevant content on cytokinin be added to the Introduction.

2) Figure 2b presents a Venn diagram of differentially expressed genes (DEGs) across different developmental stages, which is confusing. In the corresponding text (Lines 121-125), the authors do not clearly articulate the rationale for this analysis. It is recommended that the authors either clarify the purpose of this analysis in the manuscript or modify this Figure panel.

3) In Lines 268-296, the authors' presentation implies that cytokinin is involved in fruit development. However, their zeatin measurement results suggest that FvCKX1 does not act through zeatin. As is well established, biologically active cytokinins include iP, tZ, and others (Li et al., Plant Physiology, 2025; doi.org/10.1093/plphys/kiaf462). The authors only measured zeatin without distinguishing between cZ and tZ. Is such a conclusion not overly assertive? It is recommended that the authors expand the Discussion section to address this point, thereby broadening readers' understanding.

4) The authors found that FvCKX1 functions through negative regulation of ABA accumulation, an interesting finding. However, in the Discussion, the authors merely restate this result without discussing the possible mechanism by which FvCKX1, a cytokinin metabolism enzyme, regulates ABA content through FvCYP707A4a. It is recommended that the authors expand this part of the Discussion.

5) The Discussion section lacks a concluding paragraph at its end. It is recommended that the authors add one.

 

Minor points:

1) Abbreviations should be clearly defined upon first appearance in the text, and the abbreviated forms should be used consistently thereafter without alternation with full terms (e.g., ABA, GA, PCoA).

2) The manuscript mentions CKX genes from different plant species. Please add genus and species names before the gene symbols to facilitate clear identification by readers.

3) In Figures 4, 5, and 6, both letters and asterisks are used to indicate significant differences, but the figure legends do not explain whether different numbers of asterisks represent different levels of significance. This should be clarified.

4) Gene names and Fragaria vesca should be italicized throughout.

5) The Methods section lacks a description of the zeatin measurement method. It is recommended that the authors supplement this information.

Author Response

Response to Reviewer 2 Comments

We appreciate the reviewer’s valuable comments and suggestions. Please find the detailed responses below and the corresponding revisions highlighted in track changes in the re-submitted files.

Reviewer 2:

Tian et al. performed RNA-seq analysis on receptacle and achene tissues of Fragaria vesca and identified a key gene, FvCKX1, whose function was preliminarily characterized through RNAi and overexpression experiments. The manuscript is logically structured and presents relatively detailed data. However, several issues require further revision before acceptance:

Major points:

• In the Introduction, the authors discuss the roles of ethylene, auxin, and abscisic acid in fruit development, but cytokinin is not mentioned. Given that this study focuses on CKX, a cytokinin metabolic enzyme, it is recommended that relevant content on cytokinin be added to the Introduction.

Response：Thanks for the reviewer's suggestion and have now incorporated an expanded introduction regarding cytokinins in the manuscript (Lines 55-66).

• Figure 2b presents a Venn diagram of differentially expressed genes (DEGs) across different developmental stages, which is confusing. In the corresponding text (Lines 121-125), the authors do not clearly articulate the rationale for this analysis. It is recommended that the authors either clarify the purpose of this analysis in the manuscript or modify this Figure panel.

Response：We appreciate the reviewer's suggestion and have added an introduction regarding the purpose of the analysis in the main text (Lines 141-152).

• In Lines 268-296, the authors' presentation implies that cytokinin is involved in fruit development. However, their zeatin measurement results suggest that FvCKX1 does not act through zeatin. As is well established, biologically active cytokinins include iP, tZ, and others (Li et al., Plant Physiology, 2025; doi.org/10.1093/plphys/kiaf462). The authors only measured zeatin without distinguishing between cZ and tZ. Is such a conclusion not overly assertive? It is recommended that the authors expand the Discussion section to address this point, thereby broadening readers' understanding.

Response：We sincerely appreciate the reviewer's insightful comment. As correctly pointed out, cytokinins exist in diverse forms, and our measurement of only one type does not provide sufficient evidence to conclusively state that FvCKX1 does not act through zeatin. Accordingly, we have revised the relevant conclusion in the manuscript (Lines 331-332) and further discussed this point in the Discussion section (Lines 413-425) as suggested.

• The authors found that FvCKX1 functions through negative regulation of ABA accumulation, an interesting finding. However, in the Discussion, the authors merely restate this result without discussing the possible mechanism by which FvCKX1, a cytokinin metabolism enzyme, regulates ABA content through FvCYP707A4a. It is recommended that the authors expand this part of the Discussion.

Response：Following the reviewer's valuable input, we have expanded the Discussion to address the proposed regulatory mechanism whereby FvCKX1 modulates ABA levels potentially through FvCYP707A4a (Lines 426-455).

5) The Discussion section lacks a concluding paragraph at its end. It is recommended that the authors add one.

 Response：We appreciate the reviewer's suggestion and have added a concluding paragraph at the end of the Discussion section (Lines 456-468) .

Minor points:

• Abbreviations should be clearly defined upon first appearance in the text, and the abbreviated forms should be used consistently thereafter without alternation with full terms (e.g., ABA, GA, PCoA).

 Response：We appreciate the reviewer’s suggestion and have carefully reviewed and standardized all abbreviations throughout the manuscript, with necessary corrections and annotations.

• The manuscript mentions CKX genes from different plant species. Please add genus and species names before the gene symbols to facilitate clear identification by readers.

 Response：We appreciate the reviewer’s constructive suggestion and have now clearly annotated the genus and species names for all CKX genes derived from different plant species mentioned in the manuscript.

• In Figures 4, 5, and 6, both letters and asterisks are used to indicate significant differences, but the figure legends do not explain whether different numbers of asterisks represent different levels of significance. This should be clarified.

 Response：Thanks for the reviewer’s insightful comment. We have standardized the statistical analysis methods for reporting significant differences and added corresponding annotations in the figure legends.

• Gene names and Fragaria vesca should be italicized throughout.

 Response：Thanks for the reviewer’s suggestion. All gene names and Fragaria vesca (or genus/species names) are now italicized in the manuscript to comply with standard nomenclature conventions.

• The Methods sectionlacks a description of the zeatin measurement method. It is recommended that the authors supplement this information.

Response：We sincerely appreciate the reviewer's suggestion. A detailed description of the zeatin measurement method has now been added to the Methods section (Lines 483-493).。

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1. Clarity of Research Objectives

The introduction provides background on fruit development and cytokinin metabolism, but the specific research objectives and hypotheses are not clearly stated. The authors should explicitly state:

• The research gap addressed by this study
• The main hypothesis regarding FvCKX1 function
• The specific objectives of the research

This will improve the logical flow of the manuscript.

2. RNA-Seq Methodology Needs More Details

The RNA-seq methodology requires additional clarification, including:

• Criteria used for differential gene expression
• explicitly describe the software or pipelines used for transcriptomic analysis

Providing these details is essential for reproducibility and transparency.

3. Functional Validation Experiments

The RNAi and overexpression experiments are central to the conclusions of the study, but the experimental description should be improved:

• The number of biological replicates should be clearly stated.
• Details about the transformation or transient expression system should be clarified.
• Statistical analysis methods used to validate phenotypic differences should be described.
• 4. Hormonal Analysis

The manuscript suggests that FvCKX1 negatively regulates ABA synthesis, but the mechanistic explanation remains limited. The authors should:

• Provide more detailed discussion on the interaction between cytokinin metabolism and ABA signaling.
• Compare their findings with previously published studies on hormonal regulation in strawberry fruit development.

5. Discussion Needs Strengthening

The discussion section should be expanded to better interpret the biological significance of the results.

Specifically, the authors should:

• Compare their findings with other studies on cytokinin oxidase/dehydrogenase genes.
• Explain how the observed antagonistic regulation between receptacle growth and achene maturation contributes to fruit development.

Discuss possible molecular pathways involved in this regulation. I don't see a well-crafted conclusion, plz also add it briefly

For a few specific comments, see the attached file. I hope these comments will help you submit the revision

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3 Comments

We appreciate the reviewer’s valuable comments and suggestions. Please find the detailed responses below and the corresponding revisions highlighted in track changes in the re-submitted files.

Clarity of Research Objectives

The introduction provides background on fruit development and cytokinin metabolism, but the specific research objectives and hypotheses are not clearly stated. The authors should explicitly state:

The research gap addressed by this study

The main hypothesis regarding FvCKX1 function

The specific objectives of the research

This will improve the logical flow of the manuscript.

Response：We greatly appreciate the reviewers’ suggestions. We have added descriptions of these three aspects in the introduction (The research gap, Line 87-93; The main hypothesis, Line 101-102; The specific objectives, Line 90-94).

 

RNA-Seq Methodology Needs More Details

The RNA-seq methodology requires additional clarification, including:

Criteria used for differential gene expression

explicitly describe the software or pipelines used for transcriptomic analysis

Providing these details is essential for reproducibility and transparency.

Response：Thanks for the reviewers' suggestions. We have added more details of the RNA-Seq Methodology in the Methods section (Line 522-544).

 

Functional Validation Experiments

The RNAi and overexpression experiments are central to the conclusions of the study, but the experimental description should be improved:

The number of biological replicates should be clearly stated.

Details about the transformation or transient expression system should be clarified.

Statistical analysis methods used to validate phenotypic differences should be described.

Response：Thanks for the reviewers' suggestions. The number of biological replicates have been stated in figure legend (Line 254-255, line 289-290, line 367-368). Details about the transformation or transient expression system have been clarified in the methods part (Line). We have also described statistical analysis methods in the figure legend (Line 267-269, line 300-301, line 362-369)

 

 

4. Hormonal Analysis

The manuscript suggests that FvCKX1 negatively regulates ABA synthesis, but the mechanistic explanation remains limited. The authors should:

Provide more detailed discussion on the interaction between cytokinin metabolism and ABA signaling.

Compare their findings with previously published studies on hormonal regulation in strawberry fruit development.

Discussion Needs Strengthening

Response：Thanks for the reviewer’s insightful suggestion. In the Discussion section (Lines 426-455), we have further elaborated on the intrinsic mechanism by which FvCKX1 negatively regulates ABA synthesis.

 

The discussion section should be expanded to better interpret the biological significance of the results. Specifically, the authors should:

Compare their findings with other studies on cytokinin oxidase/dehydrogenase genes.

Explain how the observed antagonistic regulation between receptacle growth and achene maturation contributes to fruit development.

Discuss possible molecular pathways involved in this regulation. I don't see a well-crafted conclusion, plz also add it briefly

Response：Thanks for the reviewers’ suggestions. We have revised the discussion on the biological significance of this study, including its molecular regulatory mechanisms and biological implications.

 

For a few specific comments, see the attached file. I hope these comments will help you submit the revision

We sincerely appreciate the reviewers' constructive comments, which have significantly improved the quality of our manuscript and provided valuable guidance for future work. In response to the reviewers' concerns, we have carefully addressed each point and incorporated all suggested revisions in the revised manuscript.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript has improved in clarity and overall interpretation; however, several important issues remain only partially addressed and should be more explicitly incorporated into the text.

First, the link between FvCKX1 and ABA accumulation remains largely correlative. Although the authors acknowledge that this interaction is likely indirect and mediated through cytokinin homeostasis, the manuscript does not sufficiently emphasize the absence of direct mechanistic evidence. In particular, the distinction between FvCKX1 expression and its enzymatic activity, as well as between correlation and causation in the CK–ABA relationship, should be more clearly articulated to avoid overinterpretation.

Second, the conclusions regarding cytokinin regulation are based on limited hormonal measurements. The lack of changes in trans-zeatin is discussed, but other active cytokinin forms were not quantified. This restricts the ability to fully support the proposed role of FvCKX1 in modulating cytokinin homeostasis and should be more clearly acknowledged as a limitation.

Additionally, all functional analyses rely on transient RNAi and overexpression approaches. While the authors recognize the importance of stable transgenic lines, this limitation is not sufficiently integrated into the interpretation of the phenotypic results, which remain preliminary without stable genetic validation.

Furthermore, the proposed functional redundancy among CKX family members is presented as a likely explanation for the observed results, but no additional CKX genes were tested. This interpretation should be framed more cautiously as a hypothesis rather than a demonstrated mechanism.

Finally, the integration of cytokinin, auxin, gibberellin, and ABA pathways is well described, but remains conceptual and is not directly supported by experimental evidence in this study. This should be clearly indicated to distinguish established findings from proposed regulatory models.

Author Response

The revised manuscript has improved in clarity and overall interpretation; however, several important issues remain only partially addressed and should be more explicitly incorporated into the text.

Thanks for the reviewer’s valuable comments and suggestions. Please find the detailed responses below and the corresponding revisions highlighted in track changes in the re-submitted files.

First, the link between FvCKX1 and ABA accumulation remains largely correlative. Although the authors acknowledge that this interaction is likely indirect and mediated through cytokinin homeostasis, the manuscript does not sufficiently emphasize the absence of direct mechanistic evidence. In particular, the distinction between FvCKX1 expression and its enzymatic activity, as well as between correlation and causation in the CK–ABA relationship, should be more clearly articulated to avoid overinterpretation.

Response：Thanks for the reviewer’s valuable suggestion. FvCKX1 is a typical cytokinin oxidase/dehydrogenase whose primary function is to maintain hormonal homeostasis by degrading active cytokinins such as trans-zeatin (tZ) and isopentenyladenine (iP). We therefore propose that the regulation of ABA accumulation by FvCKX1 most likely occurs indirectly via modulation of cytokinin homeostasis, as already discussed in the manuscript (Lines 403-406). Although its core role is associated with metabolic enzyme activity, the possibility that FvCKX1 directly regulates the expression of ABA biosynthetic genes cannot be ruled out. Future studies using techniques such as ChIP-seq and DAP-seq may be required to determine whether FvCKX1 directly targets and regulates downstream genes through noncanonical functions. We have added a discussion of this potential direct regulatory mechanism to the revised manuscript (Lines 446-449). In addition, we have included a discussion distinguishing between the expression level and enzymatic activity of FvCKX1 (Lines 412-414). The crosstalk between cytokinins and ABA has also been discussed with more careful consideration.

Second, the conclusions regarding cytokinin regulation are based on limited hormonal measurements. The lack of changes in trans-zeatin is discussed, but other active cytokinin forms were not quantified. This restricts the ability to fully support the proposed role of FvCKX1 in modulating cytokinin homeostasis and should be more clearly acknowledged as a limitation.

Response：Thanks for the reviewer’s valuable suggestion. We have toned down the wording of our conclusions regarding cytokinin regulation in the manuscript, and explicitly included the measurement of other cytokinin forms as a limitation of the present study and a direction for future research in the Discussion section (Lines 461-464).

Additionally, all functional analyses rely on transient RNAi and overexpression approaches. While the authors recognize the importance of stable transgenic lines, this limitation is not sufficiently integrated into the interpretation of the phenotypic results, which remain preliminary without stable genetic validation.

Response：We appreciate the reviewer’s comment. We have toned down the description of our phenotypic conclusions, and also included the generation of stably transformed materials as a limitation of the present study and a focus for future work (Lines 461-464).

Furthermore, the proposed functional redundancy among CKX family members is presented as a likely explanation for the observed results, but no additional CKX genes were tested. This interpretation should be framed more cautiously as a hypothesis rather than a demonstrated mechanism.

Response: We appreciate the reviewer’s comment. As the reviewer pointed out, no additional CKX genes were tested. For this reason, we only provided speculation in the Discussion (Lines 409-412) and did not present this as a demonstrated mechanism.

Finally, the integration of cytokinin, auxin, gibberellin, and ABA pathways is well described, but remains conceptual and is not directly supported by experimental evidence in this study. This should be clearly indicated to distinguish established findings from proposed regulatory models.

Response：Thanks for the reviewer’s valuable suggestion. We have revised the relevant sections in the manuscript and added citations to clearly distinguish established findings from proposed regulatory models (Lines 419-427).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have answered all my concerns.

Author Response

Thanks again for the reviewers'  constructive comments and suggestions, which have significantly improved the quality of our manuscript and provided valuable guidance for future work.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have substantially improved the draft. Please make sure all the citation numbers in the text are correct.

Author Response

We have carefully checked all references and now confirm that all citation numbers in the text are correct. Thanks again for the reviewers'  constructive comments and suggestions, which have significantly improved the quality of our manuscript and provided valuable guidance for future work.