Loss of ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study is the first to uncover the non-metabolic moonlighting function of ASFT in Arabidopsis, demonstrating that ASFT negatively regulates drought tolerance by interacting with OST1 to inhibit its autophosphorylation and modulate stomatal aperture. It clarifies the upstream regulatory role of ASFT in the OST1 pathway, establishes a functional link between hydrophobic barrier synthesis and stomatal movement regulation via signal transduction, expands the molecular regulatory network upstream of OST1, and provides a novel perspective for investigating the integration of structural and signaling strategies in plant drought adaptation, thus exhibiting distinct innovation and scientific significance. However, several issues still need to be addressed and improved.

1. In Fig. 1a, the brightness of the image after Re-water is significantly lower than that of the other two images, which impairs the direct comparison of Arabidopsis phenotypic changes between the drought and re-watering stages. It is recommended to standardize the brightness of the images for the CK, Drought and Re-water treatments to ensure consistency.
2. The study performed yeast two-hybrid assays to verify the interaction between ASFT and OST1. However, the two control groups (ASFT-AD+BD and OST1-BD+AD) and serial dilutions are missing in Fig. 2a. In the absence of these two critical elements, the results of the yeast two-hybrid assays are insufficiently rigorous.
3. In lines 128–129, the sentence reads: "These were co-transformed into Agrobacterium separately with the pXY106-nYFP empty vector control, and then used to infiltrate N. benthamiana leaves". It should be noted that when the Latin name of Nicotiana benthamiana is first mentioned in the manuscript, it is inappropriate to use the abbreviation "N. benthamiana" directly. The full scientific name Nicotiana benthamiana must be used on its first occurrence.
4. In Fig. S8, the sizes of the DNA marker should be clearly labeled.
5. In Fig. 3g, the survival rate of the WT does not reach approximately 70% as shown in the bar graph of Fig. 3h; additionally, the survival rate of the ost1 mutant after rehydration is higher than that of the asft ost1 double mutant, which is inconsistent with the description in Lines 205–206.
6. The schematic diagram in Fig. 4c is overly simple. The autophosphorylation of OST1, which is a critical part of the molecular mechanism verified in this study, is not shown in the existing diagram. This makes it impossible for the schematic to fully and clearly reflect the core regulatory relationship between ASFT and OST1. It is suggested that the schematic diagram be revised to clearly illustrate the autophosphorylation process of OST1.

Author Response

Response to Reviewers

Line numbers in this response refer to the clean version of the revised manuscript.

We sincerely thank the reviewers for the valuable comments and constructive suggestions, which have helped us significantly improve the quality and clarity of our work. We have carefully considered and responded to every comment, with corresponding revisions made throughout the manuscript. Below is a point-by-point response to the reviewers' comments:

 

Comments 1: In Fig. 1a, the brightness of the image after Re-water is significantly lower than that of the other two images, which impairs the direct comparison of Arabidopsis phenotypic changes between the drought and re-watering stages. It is recommended to standardize the brightness of the images for the CK, Drought and Re-water treatments to ensure consistency.

Response 1: We sincerely thank the reviewer for pointing out the inconsistent brightness in Fig. 1a. We have now standardized the brightness and contrast across all three panels using Adobe Photoshop, ensuring that the CK, Drought, and Re-water images are visually comparable. Page 4, Line 101: The revised figure has been replaced in the manuscript.

 

Comments 2: The study performed yeast two-hybrid assays to verify the interaction between ASFT and OST1. However, the two control groups (ASFT-AD+BD and OST1-BD+AD) and serial dilutions are missing in Fig. 2a. In the absence of these two critical elements, the results of the yeast two-hybrid assays are insufficiently rigorous.

Response 2: We thank the reviewer for this critical and constructive observation. We fully agree that proper controls and serial dilutions are essential for rigorous interpretation of yeast two-hybrid results. Following this guidance, we have reperformed the yeast two-hybrid assay with the necessary controls and dilution series.

The revised Fig. 2a now includes:

1. Complete control groups:

Positive control: pGBKT7-53 + pGADT7-T (known strong interactors)

Negative control: pGBKT7-Lam + pGADT7-T (non-interacting pair)

Autoactivation controls:

AD-ASFT + BD-empty (to exclude ASFT autoactivation)

BD-OST1 + AD-empty (to exclude OST1 autoactivation)

2. Serial dilutions:

10-fold serial dilutions (10⁰, 10⁻¹, 10⁻², 10⁻³) were spotted for each transformation on:

SD/-Leu/-Trp (-LW) medium to verify co-transformation (top panels);

SD/-Leu/-Trp/-Ade/-His (-LWHA) medium supplemented with X-α-Gal and 15 mM 3-amino-1,2,4-triazole (3-AT) to suppress potential autoactivation and test for protein-protein interactions (bottom panels).

The revised Fig. 2a now provides rigorous and unambiguous evidence for the specific interaction between ASFT and OST1.

Changes in the manuscript:

Page 6, Line 161 Figure 2a: completely replaced with new Y2H data including all controls and serial dilutions.

Page 6, Lines 162-169 Figure 2 legend: updated to describe the experimental conditions.

Page 12, Lines 371-383 Materials and Methods, Section 4.4: expanded to include detailed description of controls, dilution series.

We thank the reviewer for this important suggestion, which has significantly strengthened the rigor of our yeast two-hybrid data and improved the overall quality of our manuscript.

 

Comments 3: In lines 128–129, the sentence reads: "These were co-transformed into Agrobacterium separately with the pXY106-nYFP empty vector control, and then used to infiltrate N. benthamiana leaves". It should be noted that when the Latin name of Nicotiana benthamiana is first mentioned in the manuscript, it is inappropriate to use the abbreviation "N. benthamiana" directly. The full scientific name Nicotiana benthamiana must be used on its first occurrence.

Response 3: We thank the reviewer for this careful observation regarding the proper usage of Latin binomial nomenclature. We agree that the full scientific name should be provided upon first mention, followed by the abbreviated form thereafter.

We have corrected this in the revised manuscript as follows:

Lines 129 (original): "...then used to infiltrate N. benthamiana leaves."

Lines 132 (revised): "...then used to infiltrate Nicotiana benthamiana leaves."

We have also verified the entire manuscript to ensure that all subsequent occurrences of N. benthamiana are correctly abbreviated after the first full mention.

Changes in the manuscript:

Page 5, Line 132: replaced "N. benthamiana" with "Nicotiana benthamiana".

 

Comments 4: In Fig. S8, the sizes of the DNA marker should be clearly labeled.

Response 4: We thank the reviewer for this careful observation. We agree that clearly labeled DNA marker sizes are essential for interpreting PCR genotyping results. To address this issue, we have revised Figure S8 as follows:

Added clear size indications for the DNA marker bands directly on the gel image (bp: 100, 250, 500, 750, 1000, and 2000 bp).

Updated the figure legend to include a description of the marker and the expected fragment sizes for each genotype:" The DNA marker (DL2000 Marker, Mei5bio) was run in the rightmost lane, with fragment sizes indicated on the right (bp: 100, 250, 500, 750, 1000, 2000).".

The revised Figure S8 now provides all necessary information for accurate interpretation of the genotyping results.

Changes in the manuscript:

Figure S8: replaced with revised version showing labeled DNA marker sizes

Figure S8 legend: expanded to describe marker bands and expected fragment sizes

 

Comments 5: In Fig. 3g, the survival rate of the WT does not reach approximately 70% as shown in the bar graph of Fig. 3h; additionally, the survival rate of the ost1 mutant after rehydration is higher than that of the asft ost1 double mutant, which is inconsistent with the description in Lines 205–206.

Response 5: We thank the reviewer for pointing out the discrepancies between Fig. 3g and Fig. 3h, as well as the apparent visual difference between ost1 and asft ost1 in the image.

We have re-examined the raw data from four independent experiments (20 seedlings per genotype per experiment) and made the following clarifications and corrections:

1. Discrepancy between Fig. 3g and Fig. 3h

The reviewer correctly observed that the WT survival rate in Fig. 3g appears lower than the ~70% shown in Fig. 3h. This is because each image in Fig. 3g shows only 8 representative seedlings per genotype, whereas the quantification in Fig. 3h is based on all 20 seedlings per genotype per experiment. The mean WT survival rate across four experiments was 73.75% (59/80), which is accurately reflected in Fig. 3h. To avoid confusion, we have updated the figure legend to clearly state that the images show representative seedlings, while the quantification represents the full dataset.

2. Apparent difference between ost1 and asft ost1 in Fig. 3g

The reviewer noted that ost1 appears to have a higher survival rate than asft ost1 in the image. However, statistical analysis of all four experiments (20 seedlings per genotype per experiment) shows that ost1 had a total survival of 25 out of 80 seedlings (31.25%), while asft ost1 had 28 out of 80 seedlings (35.00%). Student's t-test confirmed no significant difference between the two genotypes (P = 0.29). Therefore, the statement in Lines 205–206 ("there was no significant difference in survival between the ost1 and asft ost1 genotypes") is statistically accurate. The visual impression in the image is due to the limited number of seedlings shown (8 per genotype) and normal biological variation within a single replicate.

Changes made:

Page 8, Line 216 Figure 3g: image replaced with a more representative replicate; Lines 225-231 Figure 3 legend: updated to clarify: "Images show 8 representative seedlings per genotype; quantification in (h) represents mean ± SD of four independent experiments (20 seedlings per genotype per experiment, total n=80 per genotype).".

Lines 205–206 (original): unchanged (statistically correct).

We thank the reviewer for helping us improve the accuracy and transparency of our data presentation.

 

Comments 6: The schematic diagram in Fig. 4c is overly simple. The autophosphorylation of OST1, which is a critical part of the molecular mechanism verified in this study, is not shown in the existing diagram. This makes it impossible for the schematic to fully and clearly reflect the core regulatory relationship between ASFT and OST1. It is suggested that the schematic diagram be revised to clearly illustrate the autophosphorylation process of OST1.

Response 6: We thank the reviewer for this valuable suggestion. Following this guidance, we have revised Fig. 4c to better illustrate the autophosphorylation of OST1 and its regulation by ASFT.

The key modifications include: OST1 autophosphorylation is now clearly depicted with red "P" circles attached to OST1;

The number of "P" circles reflects the relative autophosphorylation level: three in wild-type (basal level) and four in the asft mutant (enhanced level due to loss of ASFT inhibition);

In the asft mutant panel, ASFT is shown as a gray dashed circle to indicate its absence.

These revisions make the core regulatory relationship—ASFT inhibits OST1 autophosphorylation—visually clear and accurately reflect our experimental findings.

Changes in the manuscript:

Page 9, Line 266 Figure 4c: revised as described above.

We thank the reviewer for this helpful suggestion, which has improved the clarity and impact of our schematic diagram

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1. The title is relatively lengthy. It is recommended to simplify it to "ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation."
2. The abstract section can be further refined to highlight the main findings and conclusions, for example: "We found that ASFT acts as a negative regulator of OST1, enhancing drought tolerance in Arabidopsis by restricting stomatal aperture."
3. It is suggested to include relevant research on the importance of the ABA signaling pathway and stomatal regulation in drought tolerance in the introduction section.
4. Clearly state the number of replicates for each experiment in the Materials and Methods section to increase the credibility of the results.
5. Check and update the cited literature to ensure that it includes the latest relevant research findings.
6. Simplify some complex sentences to improve the readability of the article.
7. Add an independent conclusion section at the end of the article to summarize the main findings and significance of this study.
8. Conduct a grammar and spelling check throughout the article to ensure linguistic accuracy.
9. Ensure consistent terminology usage throughout the article, for example, providing the full name "Aliphatic Suberin Feruloyl Transferase" upon its first appearance and subsequently using the abbreviation "ASFT."

Author Response

Response to Reviewers

Line numbers in this response refer to the clean version of the revised manuscript.

We sincerely thank the reviewers for the valuable comments and constructive suggestions, which have helped us significantly improve the quality and clarity of our work. We have carefully considered and responded to every comment, with corresponding revisions made throughout the manuscript. Below is a point-by-point response to the reviewers' comments:

 

Comments 1: The title is relatively lengthy. It is recommended to simplify it to "ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation."

Response 1: We thank the reviewer for the valuable suggestion to simplify the title. We agree that the original title was overly lengthy and would benefit from conciseness. However, we noted that the proposed title "ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation" could potentially introduce ambiguity, as our data demonstrate that ASFT is a negative regulator of drought tolerance—i.e., loss of ASFT enhances drought tolerance, while ASFT overexpression reduces it.

To maintain both accuracy and conciseness, we have revised the title to: Loss of ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation

This version:

Retains the core molecular mechanism (regulation of OST1 autophosphorylation);

Clearly reflects the negative regulatory role of ASFT (via "Loss of");

Is substantially more concise than the original (11 words vs. 22 words);

Avoids any potential misinterpretation of the gene's function.

We appreciate the reviewer's suggestion, which helped us craft a more precise and readable title.

 

Comments 2: The abstract section can be further refined to highlight the main findings and conclusions, for example: "We found that ASFT acts as a negative regulator of OST1, enhancing drought tolerance in Arabidopsis by restricting stomatal aperture."

Response 2: We thank the reviewer for this constructive suggestion to refine the abstract. Following this guidance, we have completely restructured the abstract to present a clear logical flow from molecular mechanism to physiological phenotype and genetic evidence, while eliminating redundancy.

Revised Abstract:

Drought stress severely constrains plant growth and productivity. To mitigate water loss, plants primarily regulate stomatal aperture through the ABA signaling pathway, where the SnRK2 family kinase OST1 acts as a central positive regulator. However, the upstream regulators that fine-tune OST1 activity remain incompletely characterized. Aliphatic Suberin Feruloyl Transferase (ASFT), a BAHD acyltransferase essential for suberin aromatic monomer biosynthesis, was previously uncharacterized regarding its function in leaves. Here, we report that ASFT negatively regulates drought tolerance in Arabidopsis by directly interacting with OST1 and inhibiting its autophosphorylation, thereby restricting stomatal aperture. Consistent with this, the asft mutant exhibited decreased water loss and enhanced survival under drought, whereas ASFT-overexpressing lines showed opposite phenotypes. BiFC, Co-IP and in vitro kinase assays confirmed that ASFT directly interacts with OST1 and suppresses its autophosphorylation, while dehydration-induced OST1 phosphorylation was elevated in the asft mutant. Genetic evidence confirmed that ASFT functions upstream of OST1. This study reveals a moonlighting role for this suberin biosynthetic enzyme in ABA signaling and provides a potential target for breeding drought-resistant crops.

Key improvements:

First mention of ASFT now includes full name with abbreviation;

Core finding presented in a single, complete sentence incorporating the reviewer's suggested phrasing;

Eliminated redundancy between mechanism statement and concluding sentence.

Changes in the manuscript:

Page 1, Lines 11-27: The Abstract has been completely revised as shown above.

We thank the reviewer for helping us craft a more focused and impactful abstract.

 

Comments 3: It is suggested to include relevant research on the importance of the ABA signaling pathway and stomatal regulation in drought tolerance in the introduction section.

Response 3: We thank the reviewer for this valuable suggestion. Following this guidance, we have revised the introduction to provide a more comprehensive overview of the ABA signaling pathway and its role in stomatal regulation. The original description of OST1 has been replaced with the following expanded text:

Original:

Open Stomata 1 (OST1), a serine/threonine protein kinase of the Sucrose Nonfermenting 1-Related Protein Kinase 2 (SnRK2) family, acts as a key hub in this pathway. Upon drought or ABA stimulation, OST1 is activated via autophosphorylation, which subsequently phosphorylates downstream targets such as ion channels and transcription factors, ultimately facilitating stomatal closure.

Revised:

Upon ABA perception, PYR/PYL receptors inhibit clade A protein phosphatase 2Cs (PP2Cs), which in turn release the inhibition of Sucrose Nonfermenting 1-Related Protein Kinase 2 (SnRK2) family kinases. Open Stomata 1 (OST1), a key SnRK2 family kinase, acts as a central regulator in this pathway. The released OST1 kinase is then activated via autophosphorylation and phosphorylates downstream targets such as anion channels, K⁺ influx channels, transcription factors and NADPH oxidases, ultimately facilitating stomatal closure.

This revised description provides a more complete picture of the ABA signaling pathway by presenting the full core cascade from PYR/PYL receptors through PP2C inhibition to SnRK2 release, clearly positioning OST1 as the key SnRK2 family kinase in this pathway. It describes the two-step activation mechanism of OST1 involving release from PP2C inhibition followed by autophosphorylation, and expands the downstream targets to include anion channels, K⁺ influx channels, transcription factors and NADPH oxidases, reflecting the multifaceted role of OST1 in guard cell signaling. Finally, it connects these molecular events to the physiological outcome of stomatal closure.

We believe this expanded background provides readers with the necessary context to fully appreciate the significance of our finding that ASFT regulates OST1 autophosphorylation, and better highlights the importance of ABA signaling and stomatal regulation in drought tolerance.

Changes in the manuscript:

Page 2, Introduction, Lines 42–48: revised as shown above.

We thank the reviewer for this constructive suggestion, which has significantly improved the depth and completeness of our introduction.

 

Comments 4: Clearly state the number of replicates for each experiment in the Materials and Methods section to increase the credibility of the results.

Response 4: We thank the reviewer for this important suggestion. Following this guidance, we have reviewed the manuscript and confirmed that the number of replicates for each experiment is already clearly stated in the corresponding figure legends. To further enhance clarity and ensure that this information is also readily available in the Materials and Methods section, we have added a new "Statistical analysis" subsection at the end of the Materials and Methods section:

"All experiments were performed with at least three independent biological replicates unless otherwise specified. The exact number of replicates for each experiment, including biological and technical replicates, is indicated in the corresponding figure legends. Data are presented as mean ± standard deviation (SD). Statistical significance between two groups was determined by two-tailed Student's t-test. Comparisons among multiple groups were analyzed by one-way ANOVA followed by appropriate post-hoc tests. A P-value < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01). All statistical analyses were performed using SPSS."

This addition ensures that readers can easily find replicate information and statistical methods in a centralized location, further supporting the credibility and reproducibility of our results.

Changes in the manuscript:

Page 13, Lines 433–441, Materials and Methods: added new "Statistical analysis" subsection at the end of the section

We thank the reviewer for helping us improve the clarity and rigor of our manuscript.

 

Comments 5: Check and update the cited literature to ensure that it includes the latest relevant research findings.

Response 5: We thank the reviewer for this valuable suggestion. Following this guidance, we have updated the reference list by adding two recent 2025 publications that are directly relevant to our study.

In the Introduction, we have added a citation to support the downstream targets of OST1 in stomatal closure: Shen et al. (2025) Proc Natl Acad Sci USA [17]. This reference provides the latest mechanistic insight into OST1-mediated activation of the SLAC1 anion channel, further supporting the central role of OST1 in stomatal regulation.

In the Discussion, we have added a citation to contextualize the moonlighting function of ASFT: Cedano et al. (2025) Int J Mol Sci [42]. This reference discusses the structural basis of protein moonlighting, which helps frame our finding that ASFT, a suberin biosynthetic enzyme, also functions as a regulator of OST1 autophosphorylation.

These additions ensure that our manuscript reflects the most current understanding of both OST1-mediated stomatal regulation and the moonlighting functions of metabolic enzymes, while maintaining the focus on our novel finding that ASFT regulates OST1 autophosphorylation.

Changes in the manuscript:

Page 2, Line 48, Introduction: added citation to Shen et al. (2025);

Page 11, Line 339, Discussion: added citation to Cedano et al. (2025);

Page 15, Lines 512–514, and Page 17, Lines 579–580 Reference section: added the two new references.

We thank the reviewer for helping us improve the timeliness of our literature citation.

 

Comments 6: Simplify some complex sentences to improve the readability of the article.

Response 6: We thank the reviewer for this valuable suggestion. Following this guidance, we have simplified several complex sentences in the manuscript to improve readability. The following are representative examples of the revisions made:

Original: This improved performance could stem from either enhanced water uptake or reduced water loss. To distinguish between these possibilities, we measured root hydraulic conductivity. Notably, the expected reduction in root suberin in the asft mutant was not accompanied by a detectable change in hydraulic conductivity compared to the wild type, implying that altered root water transport may not be a primary driver of the phenotype.

 

Revised: This improved performance could stem from either enhanced water uptake or reduced water loss. To distinguish between these possibilities, we measured root hydraulic conductivity. The asft mutant was expected to have reduced root suberin. However, its hydraulic conductivity was unchanged, indicating that altered root water transport is not the driver of its drought-tolerant phenotype.

In addition to this example, we have simplified other complex sentences throughout the manuscript by splitting long sentences, removing nested clauses, and separating multiple observations into distinct statements. All changes are highlighted in the revised manuscript for your convenience.

Changes in the manuscript:

Page 10, Introduction, Lines 298–303: revised as shown above.

We thank the reviewer for this helpful suggestion.

 

Comments 7: Add an independent conclusion section at the end of the article to summarize the main findings and significance of this study.

Response 7: We thank the reviewer for this constructive observation. We agree that an independent conclusion section helps readers quickly grasp the main findings and significance of the study. Following this suggestion, we have added a separate Conclusion section at the end of the manuscript, after the Discussion and before the References:

5. Conclusion

In this study, we identified a novel moonlighting function of ASFT in regulating drought tolerance in Arabidopsis. Our key findings demonstrate that ASFT negatively regulates drought tolerance by modulating stomatal aperture, directly interacts with OST1, and inhibits its autophosphorylation at Ser175. Genetic epistasis analysis placed ASFT upstream of OST1, as the asft ost1 double mutant exhibited phenotypes similar to the ost1 single mutant in stomatal aperture, water loss, and drought tolerance. These findings reveal a previously unrecognized regulatory mechanism in which a suberin biosynthetic enzyme moonlights as a direct inhibitor of OST1, linking metabolic processes with ABA signaling to fine-tune stomatal responses under drought stress. This work provides a potential target for breeding drought-resistant crops through genetic manipulation of ASFT expression.

This new section concisely summarizes the main findings, highlights the scientific significance, and mentions the potential application in crop breeding. We believe it improves the overall structure and readability of the manuscript.

Changes in the manuscript:

After Discussion, Page 13, Lines 442–453: added new "5. Conclusion" section

We thank the reviewer for this helpful suggestion.

 

Comments 8: Conduct a grammar and spelling check throughout the article to ensure linguistic accuracy.

Response 8: We thank the reviewer for this important suggestion. Following this guidance, we have thoroughly checked the entire manuscript for grammar and spelling errors.

Specifically, we have: Carefully reviewed each section for misspellings; Checked for consistent use of terminology throughout the text; Verified proper punctuation and sentence structure; Paid special attention to scientific terminology and gene/protein naming conventions.

All identified errors have been corrected, and the revised manuscript has been carefully proofread to ensure linguistic accuracy. The changes are highlighted in the revised manuscript for your convenience. We appreciate the reviewer's attention to this detail, which has helped improve the overall quality of our manuscript.

 

Comments 9: Ensure consistent terminology usage throughout the article, for example, providing the full name "Aliphatic Suberin Feruloyl Transferase" upon its first appearance and subsequently using the abbreviation "ASFT."

Response 9: We thank the reviewer for these constructive suggestions. Following this guidance, we have made the following revisions to the manuscript:

1. Title revision

We agree that the original title was overly lengthy. After careful consideration, we have revised the title to: Loss of ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation

This version incorporates the reviewer's suggestion for conciseness while maintaining scientific accuracy. The phrase "Loss of" is retained to accurately reflect that ASFT is a negative regulator of drought tolerance (i.e., the asft mutant exhibits enhanced tolerance, while ASFT-overexpressing lines show increased susceptibility).

2. ASFT terminology consistency

Following the reviewer's guidance on terminology consistency, we have:

Added the full name of ASFT at its first appearance in the Abstract (Lines 15-16): "Aliphatic Suberin Feruloyl Transferase (ASFT), a BAHD acyltransferase essential for suberin aromatic monomer biosynthesis..."

Removed the redundant full name from the Introduction (original Line 50), so that ASFT appears there only as the abbreviation.

Considering the title now uses the abbreviated form "ASFT", we ensured that the full name is provided at its first mention in the abstract, avoiding unnecessary repetition elsewhere.

3. Other terminology adjustments

We have also added full names for the following terms at their first appearance:

Sucrose Nonfermenting 1-Related Protein Kinase 2 (SnRK2) at Introduction, Line 44.

Escherichia coli (E. coli) at Methods, Lines 408-409.

For technical abbreviations that are standard and widely recognized (Co-IP, YFP, BiFC and GFP), we have added their full names to the Abbreviations section at the end of the manuscript, allowing readers to reference them if needed while keeping the main text concise.

Summary of changes

Title revised to "Loss of ASFT Enhances Drought Tolerance in Arabidopsis by Regulating OST1 Autophosphorylation".

ASFT full name added at Abstract, Lines 15-16 and removed from Introduction, Line 50 (original).

SnRK2 full name added at Introduction, Line 44.

Escherichia coli (E. coli) at Methods, Lines 408-409.

Full names for Co-IP, YFP, BiFC, GFP etc. added to the Abbreviations section, Page 14 Lines 477-478.

We thank the reviewer for these valuable suggestions, which have significantly improved the clarity, conciseness, and professionalism of our manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Drought leads to osmotic stress, which is accompanied by a violation of cellular metabolism and the development of oxidative stress, which ultimately has a detrimental effect on plant productivity. Therefore, it is important to develop and implement sustainable methods of agricultural production that will reduce the negative consequences of abiotic stressors and provide a solution to food security problems in the future.

One of the possible methods is the identification and study of genes (their products) associated with the resistance of plants to abiotic stresses.

In addition, it is important to study one of the adaptation mechanisms associated with stomatal movement regulation. The signal system of abscisic acid controls the functioning of the stomatal apparatus.

OST1 (Open Stomata 1), a serine/threonine protein kinase of the SnRK2 family, is an important component of this signaling pathway, which, under stress or in response to ABA, contributes to stomatal closure.

In addition to regulating the movement of the stomata, plants have hydrophobic barrier polymers (suberin-like) to limit the loss of water by cells and thus resist environmental stress. For example, aliphatic suberinferuloyltransferase (ASFT) promotes the biosynthesis of the suberin precursor.

Based on their research, the authors of the article expanded the functional aspect of ASFT and suggested that bridging the synthesis of a hydrophobic barrier with the regulation of stomatal movement by signal transduction. This fact is of interest for the integration of structural and signaling strategies in the adaptation of plants to drought.

Thus, the authors showed that ASFT (BAHD acyltransferase essential for suberin aromatic monomer biosynthesis) acts as a negative regulator of drought resistance in Arabidopsis plants. The effect is achieved by limiting the opening of the stomata by modulating the autophosphorylation of OST1 (a positive regulator of the abscisic acid signaling pathway). The authors' research made it possible to identify a potential target for the selection of drought-resistant crops.

The article corresponds to the journal's profile.

The experiments were carried out using classical analytical methods, the obtained data were statistically processed.

The work is beautifully illustrated. At least 50% of sources in References for the last 5 years.

The research is relevant, as the action of abiotic factors can have serious consequences for food security and the economy of many countries.

Among the shortcomings of the work, it is possible to note the absence of a conclusion, which slightly complicates the perception of the research results.

I believe that the study can be published after minor revision.

Author Response

Response to Reviewers

Line numbers in this response refer to the clean version of the revised manuscript.

We sincerely thank the reviewers for the valuable comments and constructive suggestions, which have helped us significantly improve the quality and clarity of our work. We have carefully considered and responded to every comment, with corresponding revisions made throughout the manuscript. Below is a point-by-point response to the reviewers' comments:

 

Comments 1: Among the shortcomings of the work, it is possible to note the absence of a conclusion, which slightly complicates the perception of the research results.

Response 1:

We thank the reviewer for this constructive observation. We agree that an independent conclusion section helps readers quickly grasp the main findings and significance of the study. Following this suggestion, we have added a separate Conclusion section at the end of the manuscript, after the Discussion and before the References:

5. Conclusion

In this study, we identified a novel moonlighting function of ASFT in regulating drought tolerance in Arabidopsis. Our key findings demonstrate that ASFT negatively regulates drought tolerance by modulating stomatal aperture, directly interacts with OST1, and inhibits its autophosphorylation at Ser175. Genetic epistasis analysis placed ASFT upstream of OST1, as the asft ost1 double mutant exhibited phenotypes similar to the ost1 single mutant in stomatal aperture, water loss, and drought tolerance. These findings reveal a previously unrecognized regulatory mechanism in which a suberin biosynthetic enzyme moonlights as a direct inhibitor of OST1, linking metabolic processes with ABA signaling to fine-tune stomatal responses under drought stress. This work provides a potential target for breeding drought-resistant crops through genetic manipulation of ASFT expression.

 

This new section concisely summarizes the main findings, highlights the scientific significance, and discusses the potential application in crop breeding. We believe it improves the overall structure and readability of the manuscript.

Changes in the manuscript:

After Discussion, Page 13, Lines 442–453: added new "5. Conclusion" section

We thank the reviewer for this helpful suggestion.

Author Response File: Author Response.docx