Glucose as a Signaling Cue Reprograms Carbon–Nitrogen–Sulfur Metabolism in Cherry Rootstock Roots

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Dear Authors,  
I noted that you have addressed all the issues I suggested. However, I recommend writing out the full names of all gene names first, followed by their abbreviations in parentheses, rather than the other way around. Genes must be in uppercase and italics. Please check this format throughout the text, specifically on lines 62-63 and 83. 

Author Response

Q: I noted that you have addressed all the issues I suggested. However, I recommend writing out the full names of all gene names first, followed by their abbreviations in parentheses, rather than the other way around. Genes must be in uppercase and italics. Please check this format throughout the text, specifically on lines 62-63 and 83. 

Response： Thank you very much for your valuable comments on our manuscript. We have carefully revised it according to your suggestions. We have standardized all gene names throughout the text. Full gene names are now written out first, followed by their abbreviations in parentheses. All gene abbreviations have been formatted in italics and uppercase. We have specifically reviewed lines 62–63 and 83, as well as the entire manuscript, to ensure consistency in format. We hope that these revisions meet your expectations. Thank you again for your time and effort in helping us improve our work.

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors performed the changes requested. The article has been improved. Therefore, article can be accepted. 

Author Response

Thank you very much for your efforts in improving our manuscript.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Glucose as a Signaling Cue Reprograms Carbon–Nitrogen–Sulfur Metabolism in Cherry Rootstock Roots

Overall, the manuscript contains interesting data and potentially important results; however, a substantial revision (major revision) is required, particularly regarding the clarity of the text, the level of methodological detail, the structure of the Results section, and a more balanced interpretation of the findings.

It is necessary that the English language is thoroughly checked, as there are many grammatical errors, repeated sentences, and several awkward formulations.

Below are detailed comments and suggested revisions according to the individual sections of the manuscript.

ABSTRACT

You list a very large number of different analyses, which makes it difficult to identify the main findings and key messages of the study. Please emphasize only the most important results and reduce the number of analyses mentioned. At the end of the abstract you make quite a strong statement considering that the study is mainly based on transcriptomic analysis. I suggest using a more cautious expression, for example suggest.

INTRODUCTION

Please revise the text so that the transitions between paragraphs are clearer, as some topics (e.g., sugar signaling and nitrogen metabolism) appear rather suddenly without a clear connection to the research problem. You also mention several physiological processes (sugar signaling, hormones, nitrogen metabolism), but it is not entirely clear which of these processes represents the main focus of the study. I suggest that at the end of the introduction you more clearly define the research hypothesis or the main research questions.

MATERIALS AND METHODS

Please check the order of the numbered subsections!!

2.1 Plant materials and glucose treatment

The description of the plant material and glucose treatment is not sufficiently detailed to allow full reproducibility of the experiment. I suggest that you further clarify the choice of glucose concentration, the method of application, and the control conditions included.

2.2 Total RNA Extraction and Sequencing

Please provide the manufacturer and catalogue number of the RNA isolation kit used. Also provide more details about the library preparation. One of the key pieces of information that is missing is the sequencing depth. In addition, was RNA quality assessed?

2.3 Filtering of Raw Data

Did you use any additional quality criteria (e.g., length trimming or removal of PCR duplicates)?

2.4 Alignment with Ribosome RNA

What proportion of reads was removed as rRNA?

2.5 Alignment to Reference Genome

Please correctly cite the web sources. In addition, I recommend describing the alignment of reads to the reference genome in more detail. It would also be useful to report the mapping rate.

2.6 The Systematic Re-annotation of Expressed Genes

The procedure for the re-annotation of expressed genes is not clearly described. Please explain in more detail the criteria used for the identification of novel genes and the method used for their functional annotation.

2.7 Quantification of Gene Abundance

Why did you use TPM as the unit of expression if differential expression was later analyzed with DESeq, which typically uses raw count data?

2.8 Principal Component Analysis

Was PCA performed on all genes or only on DEGs?

2.9 Identification of Differentially Expressed Genes

Which version of the DESeq package was used? How were the comparisons performed? Was each time point compared to T0, or were comparisons made among all time points?

2.10 Weighted Gene Co-Expression Network Analysis

Please describe and explain this analysis in more detail.

2.11 GO Enrichment Analysis

It is not clearly stated whether correction for multiple testing (e.g., FDR) was applied.

2.12 KEGG Pathway Enrichment Analysis

Which statistical method was used to calculate KEGG enrichment?

2.13 DEGs Expression Analysis by qRT-PCR

This subsection also contains far too little information. Please explain the criteria for selecting genes for validation, the number of biological and technical replicates, and how the stability of the reference gene was assessed.

RESULTS

In this section you should more clearly highlight your key results and reduce the repetition of descriptions of analyses that belong in the Materials and Methods section. In addition, in some places you explain the results too extensively, which already belongs in the Discussion section. Although you present various analyses, it would be useful to more clearly summarize the main numerical results at the beginning of each subsection, as the current presentation makes the text difficult to follow. Please also emphasize more clearly how the individual analyses complement each other.

DISCUSSION

In this section you repeat too much of what has already been written earlier. The discussion needs to be substantially revised: you should discuss your obtained results and compare them with the results of previous similar studies. This part of the manuscript is rather weak. It might also be useful to emphasize the limitations of the study (e.g., that the results are mainly based on transcriptomic analysis). More clearly highlight the practical or scientific implications of your findings for research on woody fruit species.

The manuscript repeatedly refers to the signaling role of glucose; however, exogenous glucose was used in the experiment, which can also act as a carbon source or produce osmotic effects. Therefore, it is not entirely clear how the signaling effects of glucose are distinguished from metabolic or osmotic responses. I suggest that this limitation should be more clearly addressed in the discussion.

Can you clarify whether there are experimental or literature-based data supporting the connection between the observed changes in gene expression and actual physiological changes in the roots?

Line 555–557: “WGCNA builds gene networks by leveraging the scale-free topology assumption, which helps prioritize biologically meaningful relationships. WGCNA builds….”
The same sentence appears twice. Please correct this.

Line 565: You use the phrase “isn’t just”, which is rather informal for a scientific article.

WGCNA can show correlations between genes but cannot prove regulatory or causal relationships. However, in the discussion you often speak quite deterministically, as if you have identified key regulatory mechanisms. This needs to be corrected.

CONCLUSIONS

In the conclusion you should more clearly emphasize the scientific contribution of the study and highlight what is new in your results compared to previous research. It would also be useful to briefly indicate possible directions for future research or further experimental validation of the findings.

You currently describe regulatory mechanisms of the glucose response, although the study is mainly based on transcriptomic analysis and bioinformatic interpretations. Therefore, it might be more appropriate to emphasize that the results indicate potential associations or candidate pathways that should be confirmed by further functional studies.

Comments on the Quality of English Language

It is necessary that the English language is thoroughly checked, as there are many grammatical errors, repeated sentences, and several awkward formulations.

Author Response

Q1：ABSTRACT  You list a very large number of different analyses, which makes it difficult to identify the main findings and key messages of the study. Please emphasize only the most important results and reduce the number of analyses mentioned. At the end of the abstract you make quite a strong statement considering that the study is mainly based on transcriptomic analysis. I suggest using a more cautious expression, for example suggest.

Response to Reviewer ABSTRACT

We thank the reviewer for the feedback. In response, we have revised the abstract. Specifically, we have replaced mentions of individual analyses (e.g., GO and KEGG) with more general terms such as “functional enrichment analysis” to reduce methodological detail and improve readability. While we believe the different approaches—such as DEG identification, WGCNA, and GO/KEGG—are complementary and collectively essential to addressing the biological question, we have streamlined the language to keep the focus on the biological insights. Furthermore, in line with the reviewer’s suggestion, we have toned down the conclusiveness of the final statement by replacing “demonstrate” with “suggest” to more accurately reflect the transcriptomic nature of the study.

Q2:INTRODUCTION Please revise the text so that the transitions between paragraphs are clearer, as some topics (e.g., sugar signaling and nitrogen metabolism) appear rather suddenly without a clear connection to the research problem. You also mention several physiological processes (sugar signaling, hormones, nitrogen metabolism), but it is not entirely clear which of these processes represents the main focus of the study. I suggest that at the end of the introduction you more clearly define the research hypothesis or the main research questions.

Response to Reviewer INTRODUCTION

Thank you for your insightful comments regarding the logical flow and focus of the introduction. We have carefully revised the manuscript to address your concerns as follows:

To improve the transitions between paragraphs, we have added a bridging sentence,  preventing them from appearing “suddenly”. To clarify the main focus of the study, we first acknowledge that sugar signaling intersects with various physiological processes including hormone signaling and nutrient assimilation. We then clearly state that “the primary transcriptional targets and the core metabolic pathways directly modulated by glucose in cherry roots have yet to be identified”, which establishes the research significance. Following this, we present our transcriptome analysis and report that “glucose triggers a coordinated transcriptional reprogramming of carbon, nitrogen, and sulfur metabolism, identifying these interconnected pathways as central hubs in the root response to exogenous glucose”. This structure explicitly defines our main research question—identifying the core transcriptional and metabolic targets directly modulated by glucose—and presents our key finding that carbon, nitrogen, and sulfur metabolism emerge as the central focus. We believe this revision effectively clarifies the research hypothesis and main focus of the study.

 

Q3:Please check the order of the numbered subsections!!

Response to Reviewer Comments on Materials and Methods

Thank you for pointing this out. We have carefully checked the order of the numbered subsections in the Materials and Methods and have corrected it accordingly in the revised manuscript.Thank you for your comment.

Q4: 2.1 Plant materials and glucose treatment The description of the plant material and glucose treatment is not sufficiently detailed to allow full reproducibility of the experiment. I suggest that you further clarify the choice of glucose concentration, the method of application, and the control conditions included.

Response:Thank you for your comment. We have carefully reviewed this section and confirm that the following experimental details are already clearly described in the original manuscript: Plant material: Gisela 5 seedlings, cultured on Murashige and Skoog medium for four weeks at 25°C. Growth conditions: Uniform seedlings transplanted into vermiculite, nourished with Hoagland's solution, and maintained in a growth chamber (26°C/18°C day/night, 16 h light/8 h dark). Glucose concentration and application: 0.27% glucose solution applied via irrigation. Control conditions: The 0 h time point (T0) served as the control, with samples collected immediately before treatment. Sampling time points: Lateral roots harvested at 0, 6, 12, 24, 48, and 72 h post-treatment. Sample processing: Snap-frozen in liquid nitrogen and stored at −80°C. Biological replicates: Pooled tissue from at least five seedlings per sample, with three independent replicates.

Q5: 2.2 Total RNA Extraction and Sequencing Please provide the manufacturer and catalogue number of the RNA isolation kit used. Also provide more details about the library preparation. One of the key pieces of information that is missing is the sequencing depth. In addition, was RNA quality assessed?

Response:Thank you for your suggestions. We have carefully reviewed and revised the Methods section accordingly. The descriptions of the RNA isolation kit, library preparation, sequencing depth, and RNA quality assessment have now been supplemented and clarified in the revised manuscript. We believe the current version provides sufficient detail for reproducibility. Thank you again for your valuable feedback.

Q6: 2.3 Filtering of Raw Data Did you use any additional quality criteria (e.g., length trimming or removal of PCR duplicates)?

Response:Thank you for your comment. The quality control criteria we used are clearly described in the original text: adapter removal, filtering of reads with >10% N bases, and quality-based filtering (reads with >50% bases Q≤20 were discarded). PCR duplicate removal was not performed, as it is not routinely applied in RNA-seq analysis. We believe these details are sufficient for reproducibility.

Q7: 2.4 Alignment with Ribosome RNA What proportion of reads was removed as rRNA?

Response:Thank you for your question. The proportion of reads removed as rRNA has now been provided in Supplementary Table S19. As shown in the table, rRNA-derived reads accounted for approximately 0.41% to 0.99% of clean reads across all samples, indicating effective mRNA enrichment. The remaining unmapped reads were used for subsequent genome alignment and expression quantification. We believe this additional information addresses your concern.

 

 

Q8: 2.5 Alignment to Reference Genome Please correctly cite the web sources. In addition, I recommend describing the alignment of reads to the reference genome in more detail. It would also be useful to report the mapping rate.

Response:Thank you for your suggestions. We have verified that the website link for the reference genome is correct. In addition, detailed alignment statistics, including mapping rates for each sample, have been provided in Supplementary Table S20. We believe this addresses your concern.

 

 

Q9: 2.6 The Systematic Re-annotation of Expressed Genes The procedure for the re-annotation of expressed genes is not clearly described. Please explain in more detail the criteria used for the identification of novel genes and the method used for their functional annotation.

Response:Thank you for your suggestions. We have carefully reviewed and revised this section accordingly. The description of the re-annotation procedure, including the criteria for identifying novel genes, has now been supplemented and clarified in the revised manuscript.

 

Q10: 2.7 Quantification of Gene Abundance Why did you use TPM as the unit of expression if differential expression was later analyzed with DESeq, which typically uses raw count data?

Response:Thank you for your question. We used TPM for data visualization purposes, as TPM normalizes for both gene length and sequencing depth, making it suitable for cross-sample comparisons and within-sample gene ranking. For differential expression analysis, we used raw count data as input for DESeq, which internally performs its own normalization based on the count data, as required by the DESeq statistical model . This distinction between visualization and differential analysis is standard practice in RNA-seq data analysis. 

 

Q11: 2.8 Principal Component Analysis Was PCA performed on all genes or only on DEGs?

Response:Thank you for your question. PCA was performed on all genes.

 

Q12: 2.9 Identification of Differentially Expressed Genes Which version of the DESeq package was used? How were the comparisons performed? Was each time point compared to T0, or were comparisons made among all time points?

Response:Thank you for your questions. We have supplemented the Methods section with the DESeq version (v1.20.0). Differential expression analysis was performed by comparing each time point to the T0, as well as through pairwise comparisons among all time points. We hope this addresses your concerns.

 

Q13: 2.10 Weighted Gene Co-Expression Network Analysis Please describe and explain this analysis in more detail.

Response:Thank you for your comment. We believe the current description of the WGCNA—including the soft-thresholding power (11), the scale-free topology criterion (R² > 0.8), and the use of default parameters in the blockwiseModules function—provides the core information necessary for reproducibility. We have focused on these key details to keep the Methods section concise while ensuring the analysis can be replicated. 

 

Q14: 2.11 GO Enrichment Analysis It is not clearly stated whether correction for multiple testing (e.g., FDR) was applied.

Response:Thank you for your comment. We have revised the Methods section to clarify that multiple testing correction was applied. As now stated, the p-values obtained from hypergeometric testing were adjusted using the Benjamin & Hochberg method, and GO terms with a corrected p-value ≤ 0.05 were considered significantly enriched.

 

Q15: 2.12 KEGG Pathway Enrichment Analysis Which statistical method was used to calculate KEGG enrichment?

Response:Thank you for your question. We have revised the Methods section to specify that hypergeometric testing was used to identify significantly enriched KEGG pathways. This information has now been included in the revised manuscript. 

 

Q16: 2.13 DEGs Expression Analysis by qRT-PCR This subsection also contains far too little information. Please explain the criteria for selecting genes for validation, the number of biological and technical replicates, and how the stability of the reference gene was assessed.

Response:Thank you for your comments. We have revised the Methods section to specify that eight DEGs were randomly selected for qRT-PCR validation. Three biological replicates and three technical replicates were performed for each gene. The β-tubulin gene was used as the internal reference based on its stable expression reported in previous studies.

Q17: RESULTS In this section you should more clearly highlight your key results and reduce the repetition of descriptions of analyses that belong in the Materials and Methods section. In addition, in some places you explain the results too extensively, which already belongs in the Discussion section. Although you present various analyses, it would be useful to more clearly summarize the main numerical results at the beginning of each subsection, as the current presentation makes the text difficult to follow. Please also emphasize more clearly how the individual analyses complement each other.

Response:Thank you for your valuable suggestions regarding the Results section. We have carefully revised the manuscript to present the key findings more clearly. Methodological descriptions and content not suited for the Results section have been removed, improving readability and helping readers focus on the essential points. For example, the detailed description of Gene Ontology analysis previously included in Section 3.4 has been relocated to the Methods section. Similarly, Section 3.5 has been streamlined to emphasize the key results rather than methodological details. Additional revisions have been made throughout to enhance clarity and logical flow. We believe these changes have significantly improved the Results section.

Q18:DISCUSSION In this section you repeat too much of what has already been written earlier. The discussion needs to be substantially revised: you should discuss your obtained results and compare them with the results of previous similar studies. This part of the manuscript is rather weak. It might also be useful to emphasize the limitations of the study (e.g., that the results are mainly based on transcriptomic analysis). More clearly highlight the practical or scientific implications of your findings for research on woody fruit species.The manuscript repeatedly refers to the signaling role of glucose; however, exogenous glucose was used in the experiment, which can also act as a carbon source or produce osmotic effects. Therefore, it is not entirely clear how the signaling effects of glucose are distinguished from metabolic or osmotic responses. I suggest that this limitation should be more clearly addressed in the discussion. Can you clarify whether there are experimental or literature-based data supporting the connection between the observed changes in gene expression and actual physiological changes in the roots? Line 555–557: “WGCNA builds gene networks by leveraging the scale-free topology assumption, which helps prioritize biologically meaningful relationships. WGCNA builds….” The same sentence appears twice. Please correct this. Line 565: You use the phrase “isn’t just”, which is rather informal for a scientific article. WGCNA can show correlations between genes but cannot prove regulatory or causal relationships. However, in the discussion you often speak quite deterministically, as if you have identified key regulatory mechanisms. This needs to be corrected.

 

Response:Thank you for your valuable suggestions. We have carefully revised the Discussion section to address the concerns raised. The discussion now better connects our observed transcriptional reprogramming—such as the coordinated regulation of carbon, nitrogen, and sulfur metabolism—with relevant literature on sugar signaling, nutrient use efficiency, and hormone interactions in root development. Informal language (e.g., "isn't just") has been replaced with more formal academic phrasing, and overly deterministic statements have been moderated throughout to reflect that WGCNA reveals correlations rather than causal relationships, with appropriate acknowledgment that further functional validation is needed.

Regarding the absence of morphological data, we would like to clarify that the primary objective of this study was to elucidate the immediate transcriptional response mechanisms to glucose treatment within a short-term (3-day) window, rather than to document morphological adaptations, which typically manifest over longer periods. As such, we did not systematically quantify morphological parameters. We also agree with the reviewer that exogenous glucose may exert combined signaling, metabolic, and osmotic effects. We believe these revisions have improved the scientific rigor and interpretive clarity of the manuscript. Thank you again for your constructive feedback.

 

Q19:CONCLUSIONS In the conclusion you should more clearly emphasize the scientific contribution of the study and highlight what is new in your results compared to previous research. It would also be useful to briefly indicate possible directions for future research or further experimental validation of the findings. You currently describe regulatory mechanisms of the glucose response, although the study is mainly based on transcriptomic analysis and bioinformatic interpretations. Therefore, it might be more appropriate to emphasize that the results indicate potential associations or candidate pathways that should be confirmed by further functional studies.

Response:Thank you for your thoughtful suggestions regarding the Conclusions section. We have carefully considered your feedback and would like to clarify our approach. As the Conclusions section is intended to provide a concise summary of the key contributions and main findings of the study, we have focused on highlighting the novel insights derived from our transcriptomic analysis—specifically, the integrated regulation of carbon, nitrogen, and sulfur metabolism in cherry rootstocks in response to exogenous glucose. This constitutes the primary scientific contribution of this work.Regarding the concern about describing regulatory mechanisms, we acknowledge that our findings are primarily based on transcriptomic analysis and bioinformatic interpretations. Throughout the manuscript, we have used cautious language (e.g., “suggests”, “may indicate”) to reflect that these are candidate pathways and regulatory relationships that warrant further experimental validation. As for future research directions, these are already addressed in detail within the Discussion section. We believe the Discussion is the more appropriate location for such forward-looking statements, while the Conclusions should remain focused on synthesizing the key takeaways from the current study. We hope this approach is acceptable and that our revisions adequately address your concerns.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

You have corrected it concretely and well.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this article, the authors study transcriptional changes in cherry rootstock after exogenous glucose in 6 treatments and observe changes in carbon-nitrogen-sulfur metabolism.

I have some remarks.

Indicate the meaning of the abbreviations of different genes such as GLN2, GLT1, and NRTs. Check throughout the text. 

In the introduction, it is required to discuss lateral root and rootstock formation and regulation.

Indicate the quantity of glucose required to observe the effects mentioned in the introduction, such as photosynthetic efficiency and cell proliferation.

Does adding glucose change the composition of microorganisms that live in roots, affecting their development?

Indicate the plant Gisela 5 from which they come.

The results are well written with sufficient detail. I suggest adding a figure to show the 6 root treatments established during the glucose treatment to observe some morphological changes. Also the figure 4 and 9 captions need improvement. What is the main conclusion of each figure?

The discussion needs improvement. You have a lot of data to discuss more. In essence, what are the main changes related to the different points after treatment and the effect of sulphur on root growth?

Can you suggest that glucose can be added as a fertiliser for cherry?

Author Response

Responses to Reviewer #1

Comments 1: Indicate the meaning of the abbreviations of different genes such as GLN2, GLT1, and NRTs. Check throughout the text.

Response 1: We sincerely thank the reviewer for this reminder. We have now systematically checked all gene abbreviations throughout the manuscript and have ensured that each is clearly defined at first mention. For instance, “GLN2 (glutamine synthetase 2)”, “GLT1 (glutamate synthase 1)”, and “NRTs (nitrate transporters)” are now properly introduced in the text. We believe this revision improves the clarity and accessibility of the manuscript for all readers. Thank you for highlighting this.

Comments 2: In the introduction, it is required to discuss lateral root and rootstock formation and regulation.

Response 2: Thank you for this suggestion. As suggested, we have revised the Introduction to include a more detailed discussion on the formation and regulation of lateral roots and their critical roles. 

Comments 3: Indicate the quantity of glucose required to observe the effects mentioned in the introduction, such as photosynthetic efficiency and cell proliferation.

Response 3: We appreciate the reviewer's request for clarification. The studies cited in the introduction (e.g., [12]) reported that while 0.5% exogenous glucose can significantly promote the formation of lateral roots, Excessive concentration can have an inhibitory effect. Based on this dose-dependent response, we selected a treatment concentration of 0.27% glucose for our study. This concentration was sufficient to elicit a robust transcriptional shift in our cherry rootstock system without causing acute stress, thereby allowing us to focus on the primary adaptive responses.

Comments 4: Does adding glucose change the composition of microorganisms that live in roots, affecting their development?

Response 4: We thank the reviewer for raising this interesting point regarding potential microbial involvement. Our study was specifically designed to elucidate the immediate and direct transcriptional reprogramming and physiological responses within the cherry rootstock root system itself upon glucose treatment. While changes in root-associated microbial composition are indeed a plausible and important long-term ecological consequence, they likely represent a secondary or systemic phenotypic outcome that unfolds over a different (longer) timescale. The focus of the current work is on the primary signaling and metabolic adjustments in plant tissues. The reviewer’s suggestion, however, provides an excellent direction for future research to explore the integrative plant-microbe interactions under altered carbon availability. In response, we have added a dedicated paragraph in the Discussion section to explore this idea.

Comments 5: Indicate the plant Gisela 5 from which they come.

Response 5: Thank you for this suggestion. As suggested, we have explicitly indicated the origin of the experimental materials in the revised manuscript.

Comments 6: The results are well written with sufficient detail. I suggest adding a figure to show the 6 root treatments established during the glucose treatment to observe some morphological changes. Also the figure 4 and 9 captions need improvement. What is the main conclusion of each figure?

Response 6: Regarding the suggestion to add a figure showing root treatments/morphology: We appreciate this suggestion. The primary objective of this study was to elucidate the immediate transcriptional and physiological response mechanisms to glucose treatment within a short-term (3-day) window, rather than to document morphological adaptations, which typically manifest over longer periods. As such, we did not systematically quantify morphological parameters. Figure captions: We have revised the captions for Figures 4 and 9 accordingly.

Comments 7: The discussion needs improvement. You have a lot of data to discuss more. In essence, what are the main changes related to the different points after treatment and the effect of sulphur on root growth?

Response 7: We sincerely thank the reviewer for the constructive comment. In response to the suggestion that the discussion could be further expanded to better interpret the temporal dynamics of the glucose response and the specific role of sulfur metabolism, we have substantially revised and expanded the Discussion section. Specifically, we have added new content characterizing the progressive transcriptional reprogramming observed over the treatment period, describing how the response evolves from early signaling and stress perception toward sustained metabolic reconfiguration and nutrient assimilation. Additionally, we have elaborated on the role of sulfur metabolism, emphasizing the consistent upregulation of SDI1 as a sulfur-deficiency marker and explaining how its induction under glucose treatment reflects increased demand for sulfur-containing compounds such as cysteine and glutathione, which contribute to redox homeostasis and root growth through integrated carbon-nitrogen-sulfur coordination. These additions enhance the mechanistic interpretation of our time-resolved transcriptomic data and strengthen the discussion of sulfur's role in the root response to glucose.

Comments 8: Can you suggest that glucose can be added as a fertiliser for cherry?

Response 8: We thank the reviewer for this excellent and forward-looking suggestion regarding the practical application of our findings. Indeed, the potential of glucose as a biostimulant or fertilizer supplement is an exciting translational prospect. In response, we have added a dedicated paragraph in the Discussion section to explore this idea. In this addition, we cautiously propose that, based on our observed upregulation of nutrient utilization pathways (N, C, S) and key signaling networks, controlled glucose application could theoretically be developed to modulate root system architecture and enhance nutrient use efficiency in cherry rootstocks.

 

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

After reviewing the manuscript, I would like to provide the following comments and suggestions. The methodology would benefit from further refinement, and additional experimental validation is necessary to strengthen the reported findings

L17. Please substitute hours by “h” in the abstract and in the whole manuscript.

L57-L58. Are the names of the genes the same?. Please specify the correct name of the genes. Furthermore, please include the full the name of the gene and then the acronym. i.e Auxin influx gene-1 (LUX1)

L75. Please include the full name of the genes.

 

Methods

L105. Please include if the seedlings were place on pots or in a chamber?

L110. Please substitute the “hours” by “h” as previously mentioned.

L118. Please include the amount of tissue used (0.5 mg? 1mg?).

L120. Please include the  exact or range of temperature used. (elevated temperature is subjective).

L154. I suggest that the gene co-expression network should be between the differential expressed genes with GO and KEGG terms. This will improve the results of the manuscript. Or, the authors should gave an explanation of why is not necessary create the network after the enrichment analysis.

L166. Absolute fold change ? or logartimic fold change? > 2 or >1? Please review this.

In general, Please improve the method section and explain in more detail how the analysis were conducted because several bioinformatic analysis were performed and not all are included in the method section.

Please Include how each of the figures were performed (R, tbtools, phyton?)

Any validation by QRT-PCR?

 

Results

L182. Please edit the subtitle (thistranscriptome).

L196. The authors include a Pearson heatmap correlation. However, this information is not in the method section. Please include how the heatmap was created.

L226-228. The authors state a systematic re-annotation and perform a Venn diagram. Please include this information in the method section.

L269. The legend of the figure 2B states that “DEGs defined by log2 fold change >1 “. However, in method section state “absolute fold change >2”. What is correct? Please review and edit it.

Finally, I suggest editing or changing Fig. 2B. It is hard to understand. You could use a bar chart indicating on the left the up and on the right the downregulated genes. Please consider this suggestion.

L293. WGCNA was performed using the DEGs? Do these match with the method section? Please review this.

L308. The authors state that “Progressively upregulated in response to exogenous glucose”. However, positive correlation means that two variables are in the same direction, which means a strong association. Please improve the sentence.

 

Discussion.

Based on the bioinformatic analysis, Authors need to include information about other investigations regarding the bioinformatic analysis. And then, based on this information, discuss the results and try to integrate with other investigations regarding biochemical, physiological, or gene expression experiments.

 

 

 

 

 

 

 

 

 

Author Response

Responses to Reviewer #2

Comments 1: After reviewing the manuscript, I would like to provide the following comments and suggestions. The methodology would benefit from further refinement, and additional experimental validation is necessary to strengthen the reported findings.

Response 1: We thank the reviewer for the constructive suggestions. To address the need for methodological refinement, we have thoroughly revised the Methodology section by adding a summary and supplementing key experimental and analytical details (e.g., a subsection 2.6 has been added) to ensure rigor and reproducibility. We believe these enhancements have strengthened the manuscript accordingly.

Comments 2: L17. Please substitute hours by “h” in the abstract and in the whole manuscript.

Response 2: Thank you for this suggestion.We have now revised the manuscript and have consistently used “h” as the abbreviation for “hours” throughout the text, including in the abstract. We appreciate your attention to detail, which has helped improve the consistency and professionalism of the manuscript.

Comments 3: L57-L58. Are the names of the genes the same?. Please specify the correct name of the genes. Furthermore, please include the full the name of the gene and then the acronym. i.e Auxin influx gene-1 (LUX1).

Response 3: We sincerely thank the reviewer for pointing out the  error in gene nomenclature. The intended gene symbols were AUX1/LAX1. This has been corrected in the manuscript. Furthermore, following the reviewer’s suggestion, we have now ensured that all gene/protein abbreviations mentioned in the text, including these, are clearly defined at first mention.

Comments 4: L75. Please include the full name of the genes.

Response 4: We have modified it.

Comments 5: L105. Please include if the seedlings were place on pots or in a chamber?

Response 5: We thank the reviewer for this suggestion to improve the clarity of the Materials and Methods section. In response, we have revised the text to explicitly state the cultivation environment. The sentence now reads: “For the present study, Gisela 5 seedlings were initially cultured in a tissue culture room on Murashige and Skoog medium for four weeks at 25℃.”

Comments 6: L110. Please substitute the “hours” by “h” as previously mentioned.

Response 6: Thank you for this suggestion.The correction has been made at line 116 (“hours” has been replaced with “h”). We have also performed a verification to ensure uniform usage of “h” throughout the manuscript.

Comments 7: L118. Please include the amount of tissue used (0.5 mg? 1mg?).

Response 7: Thank you for this constructive suggestion. We agree that specifying the amount of starting material is essential for reproducibility. In response, we have revised the manuscript to include the quantity of tissue used (100mg) for RNA isolation.

Comments 8: L120. Please include the exact or range of temperature used. (elevated temperature is subjective).

Response 8: Thank you for this suggestion. The fragmentation temperature has now been specified as 70°C (line 132).

Comments 9: L154. I suggest that the gene co-expression network should be between the differential expressed genes with GO and KEGG terms. This will improve the results of the manuscript. Or, the authors should gave an explanation of why is not necessary create the network after the enrichment analysis.

Response 9: Thank you for this suggestion.In the present study, we employed a parallel analytical approach in which weighted gene co‑expression network analysis (WGCNA) and functional enrichment were conducted simultaneously on the differentially expressed genes (DEGs). This design was based on the following cause: Our analysis aimed to unbiasedly and systematically reveal the transcriptional reprogramming triggered by glucose treatment. WGCNA identified three key co-expression modules from all DEGs that showed the strongest correlation with the treatment time points. Subsequent KEGG enrichment analysis of these modules revealed that they were significantly enriched in nitrogen, sulfur, and carbon metabolism pathways, respectively. To verify the robustness of this finding, we performed GO/KEGG enrichment analysis on all DEGs. The results were highly consistent, pointing to the same processes: nitrogen utilization, carbon fixation, and sulfur metabolism. This “convergence of evidence” is not redundant but rather provides strong mutual validation. This convergence supports a central conclusion: glucose coordinates a carbonnitrogensulfur metabolic network. This finding does not derive from any single analytical method but rather reflects a consistent and robust biological signal identified through multiple independent approaches, which significantly enhances the reliability of our study.

Comments 10: L166. Absolute fold change ? or logartimic fold change? > 2 or >1? Please review this.

Response 10: We thank the reviewer for this helpful comment. Accordingly, we have revised the text. The criterion has been changed from “an absolute fold change > 2” to “an absolute log₂ fold change > 1” in the revised manuscript.

Comments 11: In general, Please improve the method section and explain in more detail how the analysis were conducted because several bioinformatic analysis were performed and not all are included in the method section.

Response 11: We thank the reviewer for this important comment. We have now revised the Methods section to include details of bioinformatics analyses conducted, as suggested.

Comments 12: Please Include how each of the figures were performed (R, tbtools, phyton?)

Response 12: We thank the reviewer for the suggestion to specify the software tools used for figure generation. In response, we have thoroughly reviewed and updated the Methods section to explicitly state the software and packages used for each bioinformatics analysis and its corresponding visualization. For instance, we have clarified that the PCA was conducted using the gmodels package in R, with the resulting plot generated using the ggpubr package. Similarly, for GO enrichment visualization, we have added that the results were plotted using the ggplot2 package in R. 

Comments 13: Any validation by QRT-PCR?

Response 13: We sincerely thank the reviewer for this important suggestion. qRT-PCR validation has been performed to confirm the expression profile of 8 DEGs. The results are now included in Figure S10 and Supplementary Table S18, demonstrating significant down/up-regulation of DEGs response to glucose treatments compared to control.  We have updated the manuscript text (Lines 508–509) to reflect this validation.  

 

Supplementary Figure S10 Expression levels of DEGs determined by qRT-PCR. Asterisks indicate significant differences as determined by ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001).

 

 

Comments 14: Results, L182. Please edit the subtitle (thistranscriptome).

Response 14: We thank the reviewer for catching this error.We have revised the manuscript to correct this.

Comments 15: L196. The authors include a Pearson heatmap correlation. However, this information is not in the method section. Please include how the heatmap was created.

Response 15: Thank you for pointing out this omission. We have now included a detailed description of how the Pearson correlation heatmap was created in the Methods section.

Comments 16: L226-228. The authors state a systematic re-annotation and perform a Venn diagram. Please include this information in the method section.

Response 16: Thank you for this suggestion. We have now included the description of the systematic re-annotation and Venn diagram analysis in a new subsection (2.6) of the Materials and Methods section.

Comments 17: L269. The legend of the figure 2B states that “DEGs defined by log2 fold change >1 “. However, in method section state “absolute fold change >2”. What is correct? Please review and edit it.

Response 17: We apologize for any confusion caused by the previous discrepancy. We have now corrected and unified this criterion throughout the entire manuscript.Specifically: In the Method section (line 172), the text now reads: “an absolute log₂ fold change > 1”. In the legend of Figure 2B (lines 276-277), the text has been updated to: “with DEGs defined by |log₂ fold change| > 1”.

Comments 18: Finally, I suggest editing or changing Fig. 2B. It is hard to understand. You could use a bar chart indicating on the left the up and on the right the downregulated genes. Please consider this suggestion.

Response 18: We sincerely thank the reviewer for this constructive suggestion. Figure 2B has been completely revised into a simplified bar chart with upregulated genes displayed on the left and downregulated genes on the right. The revised figure (Figure 2B) is now included in the main manuscript, and the caption has been updated to reflect this modification.

 

Comments 19: L293. WGCNA was performed using the DEGs? Do these match with the method section? Please review this.

Response 19: We thank the reviewer for their careful reading and for raising this important methodological point. We confirm that in our analysis, WGCNA was specifically performed on the set of DEGs. We have now clarified this in both the methods. The text now explicitly states that WGCNA was conducted using “the expression matrix of all genes identified as differentially expressed (DEGs)” and in the results, we specify that “WGCNA was performed using the 10,554 DEGs”.

Comments 20: L308. The authors state that “Progressively upregulated in response to exogenous glucose”. However, positive correlation means that two variables are in the same direction, which means a strong association. Please improve the sentence.

Response 20: We apologize for the lack of clarity in the original manuscript. The description of “Progressively upregulated in response to exogenous glucose” was based on the Pearson correlation coefficient in Figure 3B. We have now revised the text to explicitly reference the figure and clarify the basis for our conclusion, ensuring accurate interpretation.

Comments 21: Based on the bioinformatic analysis, Authors need to include information about other investigations regarding the bioinformatic analysis. And then, based on this information, discuss the results and try to integrate with other investigations regarding biochemical, physiological, or gene expression experiments.

Response 21: Thank you for this suggestion. In response, we have substantially expanded the Discussion section. Specifically, the revised discussion now connects our observed transcriptional reprogramming - such as the coordinated regulation of carbon, nitrogen, and sulfur metabolism and the sustained induction of stress-responsive and nutrient assimilation pathways - with relevant experimental evidence on sugar signaling, nutrient use efficiency, redox homeostasis, and hormone interactions in root development. These additions provide a more comprehensive and integrative interpretation of our data.

 

 

 

 

 

 

 

Author Response File: Author Response.docx