Analysis of Axillary Bud Germination Regulatory Network in Sugarcane Based on Transcriptome and Weighted Gene Co-Expression Network Analysis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

A well-written article. Easy to follow. Easy to read. The pictures and illustrations are appropriate. The results are well presented.

Author Response

We sincerely thank the reviewer for the positive assessment and encouraging comments on our work. We are delighted to hear that the manuscript is well written, easy to understand, and accessible to readers. We also appreciate the reviewer's positive feedback regarding the appropriateness of the figures and illustrations, as well as the clear presentation of the results. Such constructive recognition from an expert in the field is highly encouraging for us. We hope the revised manuscript meets the reviewer's expectations.

Reviewer 2 Report

Comments and Suggestions for Authors

This research is very interesting and relevant. The scientific world needs such research. However, there are very serious shortcomings in the presentation of this study:

1. The Introduction chapter very sparingly describes the experience of analyzing the genetic component in axillary bud formation in other crops. Overall, the global experience in this chapter is presented by a very small number of sources and seems incomplete and unrepresentative.
2. The Materials and Methods chapter does not specifically describe the characteristics of the selected genotype. Where did it come from? How many plants were involved, 15 or fewer? Were segments taken from a single mother plant or randomly from a sample? What was the lighting condition at each stage?
3. Chapter 4.2: How were extracts prepared for chromatography?
4. General note regarding the Materials and Methods chapter: What instruments were used for the analyses?
5. For clarity, Table 3 should indicate which signaling pathway the selected gene regions correspond to (IAA, CTA, ABA, or GA).
6. The "Discussion" chapter appears unfinished. The results are numerous and very interesting, but this chapter lacks any analysis of the data obtained, especially with respect to each other. Gene expression and hormone levels are a very interesting and well-studied topic, but this study lacks an in-depth analysis and fails to take into account the extensive experience of other researchers. Furthermore, the comparison presented with global experience relies solely on a single point of view, which is unacceptable when analyzing results of this level.
7. The data and captions for some figures are illegible: Fig. 2B, Fig. 6D.
8. The "Conclusions" chapter should specifically present the conclusions of the study, not a list of what you did.

Author Response

Comments 1: [The Introduction chapter very sparingly describes the experience of analyzing the genetic component in axillary bud formation in other crops. Overall, the global experience in this chapter is presented by a very small number of sources and seems incomplete and unrepresentative.

Response 1: In model plants and other cereals, the genetic framework governing axillary bud outgrowth has been extensively studied. For instance, genes such as TEOSINTE BRANCHED1 (TB1) in maize and its rice ortholog FINE CULM1 (FC1) act as key integrators of strigolactone signaling to repress tillering [6-8]. The MONOCULM1 (MOC1) gene in rice is essential for axillary bud formation [9,10], while the MORE AXILLARY GROWTH (MAX) pathway in Arabidopsis defines the strigolactone biosynthesis and signaling cascade [11,12]. These studies have established that hormone crosstalk, particularly among auxin, cytokinin, and strigolactones—forms the core of the regulatory network controlling shoot branching. In crops, the extent of axillary bud outgrowth directly determines plant architecture and final yield components such as tiller or branch number [13,14]. In sugarcane, the germination of axillary buds is a critical agronomic trait that significantly impacts plant architecture, ratoon performance, and effective stalk numbers and cane yield [3]. Previous studies in sugarcane have indicated that hormones such as auxin and cytokinin are major signaling molecules affecting this process [15-17]. At the molecular level, the high expression of strigolactone pathway-related genes (e.g., ScHTD2, ScF-box, ScD27) has been found to inhibit axillary bud germination [18-20], while the SoMADS57 gene may positively regulate tillering by suppressing this pathway [21]. These findings suggest that the core hormonal regulators identified in model plants are also relevant in sugarcane. However, the dynamic changes and synergistic mechanisms of multiple hormones, particularly gibberellin (GA), abscisic acid (ABA), and ethylene (ETH), whose roles in this process remain elusive—during the precise timing of axillary bud germination in sugarcane are still poorly understood. Therefore, a systematic investigation integrating hormone dynamics with gene expression profiles is urgently needed to elucidate the regulatory network underlying this key agronomic trait in this complex polyploid crop. (pp.2, lines 54–78)

Thank you for pointing this out, we agree with this comment. Therefore, I have added insights from genetic analyses of axillary bud formation in model plants and other crops and made corresponding revisions to the logical flow of the original second paragraph. I have also removed references [6] and [9]. (pp.2, lines 54–78)

 

Comments 2: The Materials and Methods chapter does not specifically describe the characteristics of the selected genotype. Where did it come from? How many plants were involved, 15 or fewer? Were segments taken from a single mother plant or randomly from a sample? What was the lighting condition at each stage?

Response 2: The commercial sugarcane variety XTT22 was used in this study. Plants were grown under standard agronomic practices in the experimental fields of the Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, Kaiyuan, China (23°42′N, 103°16′E). Healthy, 320-day-old (mature stage) field-grown plants were selected for sampling. To induce axillary bud germination, stem segments (approximately 8 cm in length) containing a single intact axillary bud were excised from the middle to upper portions of 20 randomly selected mother plants to account for biological variation. After surface sterilization in 1% carbendazim solution (Hangzhou JiaBiQi Biotechnology Co., Ltd., Hangzhou, China) for 5 min followed by three rinses with sterile distilled water, the segments were placed horizontally on trays lined with three layers of moist sterile gauze. Germination was induced in a constant-temperature incubator (MGC-350HP-2, Shanghai Hengke Scientific Instrument Co., Ltd., Shanghai, China) at 28 °C with 70% relative humidity in the dark. Axillary bud tissues were collected at five developmental stages defined by morphological changes: dormancy (XM, bud not swollen), initiation (MD, bud tip showing green coloration), swelling (PD, bud body visibly enlarged), elongation (SC, bud body elongated), and first-new-leaf stage (YY, new leaf visible at bud tip). At each stage, approximately 20 buds were pooled to form one biological replicate, and three independent biological replicates were collected. All samples were immediately frozen in liquid nitrogen and stored at −80°C until use for transcriptome sequencing and hormone analysis.. (pp.14-15, lines325-344)

Thank you for pointing this out; we agree with your comment. On page 14, in the first paragraph of Section 4.1, “Plant Materials and Processing,” I have provided an explanation and additional details regarding the issues you raised, such as the source of the sugarcane and the conditions for inducing axillary bud germination.

 

Comments 3: Chapter 4.2: How were extracts prepared for chromatography?

Response 3: The contents of IAA, GA, ABA, and CTK were determined using high-performance liquid chromatography (HPLC) following the method described by Gao et al. [40]. HPLC conditions: analyses were performed on a Rigol L3000 HPLC system (Rigol Technologies, Beijing, China) equipped with a Kromasil C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol and 1% acetic acid aqueous solution (30:70, v/v) at a flow rate of 0.8 mL/min. The injection volume was 10 μL, the column temperature was maintained at 30°C, and the detection wavelength was set at 254 nm with a run time of 50 min. The column was equilibrated with the mobile phase until a stable baseline was achieved before sample injection. Hormone extraction: Frozen bud samples (approximately 0.4 g) were ground in a mortar and extracted overnight at 4°C with 1 mL of pre-cooled methanol: water: acetic acid (80:20:1,v/v/v). The homogenate was centrifuged at 8000× g for 10 min. The residue was re-extracted with 0.5 mL of the same solvent for 2 h and centrifuged again. The supernatants were combined and evaporated to dryness under a nitrogen stream at 40°C. The residue was decolorized three times with 0.5 mL of petroleum ether, and the upper ether phase was discarded. The lower aqueous phase was adjusted to pH 2.8 with saturated citric acid solution and extracted three times with equal volumes of ethyl acetate. The organic phase was collected, evaporated to dryness under nitrogen, and redissolved in 0.5 mL of methanol. The solution was filtered through a syringe filter into a sample vial with an insert for HPLC analysis. Ethylene (ETH) content was determined according to the method described by Wang et al..[41], using a Thermo Scientific Trace 1310 gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA). Three replicates were performed per sample. Analyses were performed on a gas chromatograph equipped with a hydrogen flame ionization detector. The injector temperature was set at 60°C, the detector temperature at 150°C, and the column oven temperature at 60°C. After ignition and baseline stabilization for 30 min, samples were injected for analysis. (pp.15, lines 346–371)

Thank you for pointing this out, we agree with this comment. We have accordingly added detailed information on experimental parameters and instruments in Section 4.2 (Measurement of Endogenous Hormones) (pp.15, lines 346–371).

 

Comments 4: General note regarding the Materials and Methods chapter: What instruments were used for the analyses?

Response 4:

1. constant-temperature incubator (Shanghai Hengke Scientific Instruments Co., Ltd., Shanghai, China) (pp. 14, lines 355–356)
2. Rigol L3000 HPLC system(Rigol Technologies, Beijing, China) (pp. 15, lines 348–349)
3. Thermo Scientific Trace 1310 gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) (pp. 15, lines 365–366)
4. ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) (pp. 16, lines 404)
5. Prime Script™ II 1st Strand cDNA Synthesis Kit (TaKaRa, China) (pp. 15, lines 375).
6. (Vazyme Biotech Co., Ltd., Nanjing, China) (pp. 15, lines 379–380). (pp. 16, lines 405)
7. Langfelder, P.; Horvath, S. WGCNA: an R package for weighted correlation network analysis.BMC Bioinformatics 2008, 9, 559, doi:10.1186/1471-2105-9-559. (pp. 16, lines 393)

Thank you for pointing this out. In the revised manuscript, we have specified the instruments used for each analysis throughout the Materials and Methods section. All instruments are now described with their model numbers, manufacturers, and locations. In addition, we have added a citation for the WGCNA [Langfelder and Horvath, 2008] in Section 4.5 (Weighted Gene Co-expression Network Analysis) to provide the appropriate methodological reference.

 

Comments 5: For clarity, Table 3 should indicate which signaling pathway the selected gene regions correspond to (IAA, CTA, ABA, or GA).

Response 5: To validate the reliability of the transcriptome data, six DEGs involved in the IAA, CTK, and ABA signaling pathways were selected for qRT-PCR analysis. Results showed (Figure.8) that ScAUX1 (Cluster-9372.56238, IAA pathway) and ScB-ARR (Cluster-9372.49666, CTK pathway) exhibited upregulation followed by downregulation during axillary bud emergence. Conversely, ScIAA9 (Cluster-17038.0, IAA pathway) and ScSnRK2 (Cluster-9372.40320, ABA pathway), and ScPYL (Cluster-9372.42029, ABA pathway) were downregulated, while ScA-ARR (Cluster-9372.36004, CTK pathway) was upregulated. These findings are highly concord with RNA-seq results, confirming the reliability of the transcriptome sequencing data. (pp. 12, lines 227–231)

We thank the reviewer for this suggestion. To improve clarity, we have now explicitly indicated the corresponding hormone signaling pathway for each gene in the main text (Section 2.7, pp. 12, lines 227–231) by adding the pathway information in parentheses after the gene names (e.g., ScAUX1 (Cluster-9372.56238, IAA pathway)). This allows readers to easily identify the pathway association without expanding Table 3.

 

Comments 6: The "Discussion" chapter appears unfinished. The results are numerous and very interesting, but this chapter lacks any analysis of the data obtained, especially with respect to each other. Gene expression and hormone levels are a very interesting and well-studied topic, but this study lacks an in-depth analysis and fails to take into account the extensive experience of other researchers. Furthermore, the comparison presented with global experience relies solely on a single point of view, which is unacceptable when analyzing results of this level.

Comments 6: In this study, IAA and GA contents decreased continuously and significantly throughout the germination process, suggesting their dominant roles in maintaining bud dormancy. Conversely, the peak accumulation of CTK (6-BA) at the initial stage of germination, together with the decline in ABA content during the swelling stage, synergistically constituted a "germination initiation signal". The subsequent increase in ETH content during the late stage of germination may be involved in organ differentiation. These results indicate that axillary bud germination is not determined by a single hormone but rather depends on the precise switching of antagonistic hormone ratios—such as IAA/CTK and GA/ABA—at specific developmental nodes.

The coordinated changes in auxin signaling components further support this model. GH3 family genes, which encode IAA-conjugating enzymes that inactivate free IAA [26], showed sustained downregulation throughout germination, indicating reduced conversion of active IAA to inactive conjugates. However, this feedback mechanism did not prevent the overall decline in IAA content, suggesting that reduced auxin biosynthesis or enhanced degradation is the primary cause. The expression of AUX/IAA genes was largely downregulated, particularly at later stages, which may relieve the transcriptional repression of ARF factors and promote bud outgrowth [27]. Collectively, these expression patterns are consistent with the established role of auxin as a negative regulator of axillary bud outgrowth [28,29].

Conversely, the dynamics of CTK content were tightly associated with the expression of its downstream signaling genes. Type-A ARR genes, which are primary cytokinin response regulators rapidly induced by cytokinin treatment in Arabidopsis [30] showed sustained upregulation during the early stages when CTK levels peaked (MD and PD) (Figure 5B). In rice, similar induction of OsRR genes occurs upon cytokinin application, and elevated cytokinin levels promote tiller bud outgrowth by counteracting auxin-mediated inhibition [31]. Importantly, the peak of CTK accumulation occurred earlier than the significant decline in IAA content, supporting the established antagonistic interaction between cytokinin and auxin in shoot branching regulation [32,33]. This temporal hierarchy suggests that early CTK accumulation serves as a key leading event that initiates downstream signaling and overcomes IAA-mediated inhibition. These findings provide direct theoretical targets for promoting early and vigorous growth in sugarcane through exogenous hormone regulation, such as foliar application of CTK or GA inhibitors at the seedling stage. (pp. 12–13, lines 239–272)

We thank the reviewer for this valuable suggestion. We agree that the original Discussion section did not adequately integrate the multiple datasets or place our findings within the broader context of existing literature. We have now substantially expanded the Discussion to provide a more in-depth analysis of the relationship between hormone dynamics and gene expression patterns, while incorporating a broader range of studies from model plants and other crops. Two previously cited references [21,22] were removed due to reduced relevance after rewriting, but we have added eight new citations in the revised Discussion. These revisions are marked in red in the revised manuscript (pp. 12–13, lines 239–272).

 

Comments 7: The data and captions for some figures are illegible: Fig. 2B, Fig. 6D.

Response 7:

Fig.2B (pp. 5, lines 130)

Fig. 6D (pp. 10, lines 196)

Thank you for pointing this out. The revised Fig.2B and Fig.6D have been updated on in the revised manuscript. We confirm that all data points and captions are now clearly legible.

 

Comments 8: The "Conclusions" chapter should specifically present the conclusions of the study, not a list of what you did.

Response 8: This study provides a systematic characterization of the hormonal and transcriptional dynamics governing axillary bud germination in sugarcane. Our results reveal that axillary bud outgrowth is associated with a coordinated decline in IAA and GA levels, coupled with a transient accumulation of CTK at the initiation stage, suggesting that a hormonal switch involving antagonistic hormone ratios—particularly IAA/CTK and GA/ABA is critical for dormancy release. Transcriptomic analysis further demonstrates that this hormonal shift triggers extensive reprogramming of gene expression, with differentially expressed genes enriched in pathways related to hormone signal transduction, starch and sucrose metabolism, and photosynthesis, reflecting the transition from heterotrophic dormancy to autotrophic growth. Through WGCNA, we identified two key co-expression modules (antiquewhite4 and darkorange2) that are significantly correlated with hormone dynamics. Seven hub transcription factors, including ScTCP5, ScNAC019, and the conserved ScSCR-ScSHR1 regulatory module, were identified as central nodes linking hormone signaling to downstream developmental programs. Notably, the presence of the SHR-SCR module in axillary buds suggests a conserved mechanism for meristem maintenance across different organs. Collectively, these findings establish a foundation for understanding the molecular regulatory network underlying axillary bud germination in sugarcane and provide candidate gene resources for molecular breeding aimed at improving tillering and yield in this important crop. (pp.17, lines 420-438)

We thank the reviewer for this valuable suggestion. We have thoroughly revised the Conclusions section to focus on the key findings and scientific implications of our study, rather than summarizing the experimental procedures. The revised section now presents the main conclusions drawn from our results, including the hormonal dynamics, the identification of key co-expression modules, and the potential roles of hub transcription factors in regulating axillary bud germination in sugarcane.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is devoted to the study of hormonal and transcriptional dynamics underlying the germination of axillary buds in sugarcane. This is a very interesting and actual topic, because axillary bud germination is a critical agronomic trait that directly determines seedling emergence, tillering capacity, and ultimately, stalk number and cane yield. According to the authors, these findings not only deepen understanding of the molecular mechanisms underlying axillary bud germination in sugarcane but also provide a solid theoretical foundation and genetic resources for the molecular breeding of new sugarcane varieties with high yield and high sucrose content. However, to improve the quality of the manuscript, I propose the following revisions.

1. Please do not use abbreviations in the manuscript title.
2. Most keywords duplicate words in the manuscript title.
3. Overall, the Materials and Methods section is very brief. I have several questions regarding sections 4.1 and 4.2:

- What does the term "mature sugarcane plants" mean? How many days did these plants grow? Where were these plants grown (field, greenhouse);

- How was axillary bud germination induced;

- How was axillary bud tissue collected;

- How were hormones extracted from plant tissues.

4. The style of several sentences (lines 297-299 and 316-317) should be revised. It reads more like instructions than a description of the methods.
5. A separate Statistical Analysis subsection should be created, and information about the correlation coefficient, the PCA program, and the PCA model should be added.
6. Lines 92-93. Inconsistency between the text and the data in Figure 1C. In this figure, the 6-BA (CTK) content at the SC and YY stages did not differ significantly from the XM stage.

7. Rows 96-99. Inconsistency between the text and the data in Figure 1F. In this figure, the ETH content at the MD stage was higher than at the XM stage.

Author Response

Comment 1: Please do not use abbreviations in the title.

Response1: Thank you for pointing this out, we agree with this comment.We have expanded the abbreviation "WGCNA" to its full name "Weighted Gene Co-expression Network Analysis" in the title.

 

Comments 2: Most keywords overlap with the words in the title.

Response 2: Thank you for pointing this out, we agree with this comment.We have revised the keywords to avoid repetition with the title. We changed the keyword from sugarcane (Saccharum spp.); axillary bud; germination; transcriptome; plant hormone; weighted gene co-expression network analysis (WGCNA) to sugarcane (Saccharum spp.); axillary bud; transcriptome; hormonal crosstalk; tillering; gene regulatory network(lines:41-42)

 

Comments 3: Overall, the Materials and Methods section is very brief. I have several questions regarding sections 4.1 and 4.2:

- What does the term "mature sugarcane plants" mean? How many days did these plants grow? Where were these plants grown (field, greenhouse).

- How was axillary bud germination induced.

- How was axillary bud tissue collected.

- How were hormones extracted from plant tissues.

Response 3:

4.1. Plant Materials and Processing

The commercial sugarcane variety XTT22 was used in this study. Plants were grown under standard agronomic practices in the experimental fields of the Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, Kaiyuan, China (23°42′N, 103°16′E). Healthy, 320-day-old (mature stage) field-grown plants were selected for sampling. To induce axillary bud germination, stem segments (approximately 8 cm in length) containing a single intact axillary bud were excised from the middle to upper portions of 20 randomly selected mother plants to account for biological variation. After surface sterilization in 1% (w/v) carbendazim solution (Hangzhou JiaBiQi Biotechnology Co., Ltd., Hangzhou, China) for 5 min followed by three rinses with sterile distilled water, the segments were placed horizontally on trays lined with three layers of moist sterile gauze. Germination was induced in a constant-temperature incubator (MGC-350HP-2, Shanghai Hengke Scientific Instrument Co., Ltd., Shanghai, China) at 28°C with 70% relative humidity in the dark. Axillary bud tissues were collected at five developmental stages defined by morphological changes: dormancy (XM, bud not swollen), initiation (MD, bud tip showing green coloration), swelling (PD, bud body visibly enlarged), elongation (SC, bud body elongated), and first-new-leaf stage (YY, new leaf visible at bud tip). At each stage, approximately 20 buds were pooled to form one biological replicate, and three independent biological replicates were collected. All samples were immediately frozen in liquid nitrogen and stored at −80°C use for transcriptome sequencing and hormone analysis. (pp. 14, lines 325–344)

4.2 Measurement of Endogenous Hormones

The contents of IAA, GA, ABA, and CTK were determined using high-performance liquid chromatography (HPLC) following the method described by Gao et al. [40]. HPLC conditions: analyses were performed on a Rigol L3000 HPLC system (Rigol Technologies, Beijing, China) equipped with a Kromasil C18 reversed-phase column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol and 1% acetic acid aqueous solution (30:70, v/v) at a flow rate of 0.8 mL/min. The injection volume was 10 μL, the column temperature was maintained at 30°C, and the detection wavelength was set at 254 nm with a run time of 50 min. The column was equilibrated with the mobile phase until a stable baseline was achieved before sample injection. Hormone extraction: Frozen bud samples (approximately 0.4 g) were ground in a mortar and extracted overnight at 4°C with 1 mL of pre-cooled methanol: water: acetic acid (80:20:1,v/v/v). The homogenate was centrifuged at 8000× g for 10 min. The residue was re-extracted with 0.5 mL of the same solvent for 2 h and centrifuged again. The supernatants were combined and evaporated to dryness under a nitrogen stream at 40°C. The residue was decolorized three times with 0.5 mL of petroleum ether, and the upper ether phase was discarded. The lower aqueous phase was adjusted to pH 2.8 with saturated citric acid solution and extracted three times with equal volumes of ethyl acetate. The organic phase was collected, evaporated to dryness under nitrogen, and redissolved in 0.5 mL of methanol. The solution was filtered through a syringe filter into a sample vial with an insert for HPLC analysis. Ethylene (ETH) content was determined according to the method described by Wang et al..[41], using a Thermo Scientific Trace 1310 gas chromatograph (Thermo Fisher Scientific, Waltham, MA, USA). Three replicates were performed per sample. Analyses were performed on a gas chromatograph equipped with a hydrogen flame ionization detector. The injector temperature was set at 60°C, the detector temperature at 150°C, and the column oven temperature at 60°C. After ignition and baseline stabilization for 30 min, samples were injected for analysis. (pp. 15, lines 346–371).

Thank you for pointing this out; we agree with your comments. In Section 4.1 (Plant Materials and Processing), we have addressed the issues raised by providing additional explanatory details, including plant age, growth conditions, sampling strategy, and germination induction (pp. 14-15, lines 325–344). In Section 4.2 (Measurement of Endogenous Hormones), we have added detailed information on experimental parameters and instruments (pp. 15, lines 346–371). These revisions are marked in red in the revised manuscript. We believe these additions significantly improve the clarity and reproducibility of the experimental procedures.

Comments 4: The style of several sentences (lines 297-299 and 316-317) should be revised. It reads more like instructions than a description of the methods.

Response 4:

1.For lines 297-299

To induce axillary bud germination, stem segments (approximately 8 cm in length) containing a single intact axillary bud were excised from the middle to upper portions of 20 randomly selected mother plants to account for biological variation. After surface sterilization in 1% carbendazim solution (Hangzhou JiaBiQi Biotechnology Co., Ltd., Hangzhou, China) for 5 min followed by three rinses with sterile distilled water, the segments were placed horizontally on trays lined with three layers of moist sterile gauze. Germination was induced in a constant-temperature incubator (MGC-350HP-2, Shanghai Hengke Scientific Instrument Co., Ltd., Shanghai, China) at 28°C with 70% relative humidity in the dark. (pp. 14, lines 329-337)

2. For lines 316-317

In Section 4.2 (Measurement of Endogenous Hormones), we have supplemented the detailed content and revised the text to avoid reading more like instructions than a description of the methods.

(pp. 15, lines 346-371)

Thank you for pointing this out; we agree with your comment. We have revised the indicated sentences to adopt a descriptive style appropriate for the Materials and Methods section. These revisions are marked in red in the revised manuscript.

 

Comments 5: A separate Statistical Analysis subsection should be created, and information about the correlation coefficient, the PCA program, and the PCA model should be added.

Response 5: Hormone data and qRT-PCR data were analyzed using Microsoft Excel 2022 (Microsoft Corp., Redmond, WA, USA) and GraphPad Prism 9.5 (GraphPad Software, San Diego, CA, USA) and expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Tukey’s post hoc test was performed using GraphPad Prism 9.5, and statistical significance was defined as P < 0.05. PCA analyses was employed R 4.1.0 (R Foundation for Statistical Computing, Vienna, Austria) (pp.17, lines 313–418)

Thank you for pointing this out; we agree with your comment. We have merged Section 4.7 with the PCA analysis and revised it as "4.7 Statistical Analysis" (pp.17, lines 313–418), incorporating detailed information on statistical methods, PCA procedures, and correlation analyses. The changes are marked in red.

 

Comments 6: Lines 92-93. Inconsistency between the text and the data in Figure 1C. In this figure, the 6-BA (CTK) content at the SC and YY stages did not differ significantly from the XM stage.

Response 6: During the early stages of bud germination (MD and PD), 6-BA (CTK) content rapidly increased to a peak value of 1.44 μg/g FW. However, at the later stages (SC and YY), CTK levels were not significantly different from those at the dormancy stage (XM) (Figure 1C). These results suggest that CTK plays a key promoting role in the initiation of bud germination. (pp. 3, lines 99–104).

Thank you for pointing this out; we agree with your comment. We have revised the description of CTK dynamics in the Results section to accurately reflect the data presented in Figure 1C. The revised text now states that CTK levels increased during the early stages of bud germination (MD and PD) but showed no significant difference at the later stages (SC and YY) compared with the dormancy stage (XM). This correction is marked in red in the revised manuscript (pp. 3, lines 99–104).

 

Comments 7: Rows 96-99. Inconsistency between the text and the data in Figure 1F. In this figure, the ETH content at the MD stage was higher than at the XM stage.

Response 7: ETH content increased transiently at the initiation stage (MD), decreased during the swelling and elongation stages (PD and SC), and then significantly rebounded during the first-new-leaf stage (YY) (Figure 1F). (pp. 3, lines 106-109).

Thank you for pointing this out; we agree with your comment. We have revised the description of ETH dynamics in the Results section to accurately reflect the data in Figure 1F. The revised text now indicates that ETH content increased transiently at the initiation stage (MD) before decreasing during the swelling and elongation stages (PD and SC), followed by a significant rebound at the first-new-leaf stage (YY). This correction is marked in red in the revised manuscript (pp. 3, lines 106-109).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks to the authors for carefully revising the manuscript. The study has become more presentable and understandable. However, a few minor issues remain:
1. The Abstract section is too cumbersome; I recommend shortening it and making it more concise.
2. Keyword terms are generally not duplicated in the title.
3. Please check the text for typos and errors.

Comments on the Quality of English Language

Thanks to the authors for carefully revising the manuscript. The study has become more presentable and understandable. However, a few minor issues remain:
1. The Abstract section is too cumbersome; I recommend shortening it and making it more concise.
2. Keyword terms are generally not duplicated in the title.
3. Please check the text for typos and errors.

Author Response

Comments 1: The Abstract section is too cumbersome; I recommend shortening it and making it more concise.

Response 1: Axillary bud germination in sugarcane is a critical agronomic trait that directly determines seedling emergence and tillering capacity; however, its molecular regulatory mechanisms remain poorly understood. In this study, we systematically investigated the hormonal dynamics and transcriptomic profiles of the sugarcane cultivar XTT22 across five developmental stages (from dormancy to the first-new-leaf stage). Our results revealed that abscisic acid (ABA) content fluctuated during germination, whereas indole-3-acetic acid (IAA) and gibberellin (GA) levels decreased significantly, suggesting their negative regulatory roles. In contrast, cytokinin (CTK) and ethylene (ETH) contents increased at the initiation stage, indicating positive promoting functions. Transcriptome analysis identified 31,513 differentially expressed genes (DEGs), which were significantly enriched in pathways related to hormone signal transduction, starch/sucrose metabolism, and photosynthesis. Weighted gene co-expression network analysis (WGCNA) constructed 12 co-expression modules, among which the antiquewhite4 module (negatively correlated with IAA, GA, and ABA contents) and the darkorange2 module (positively correlated with cytokinin content) were identified as key regulatory modules. From these modules, seven core hub transcription factors (e.g., ScTCP5, ScSCR, and ScSHR1) were screened and their expression patterns were validated by RT-qPCR. Furthermore, the expression trends of six hormone-related DEGs were highly consistent with the RNA-seq data. Collectively, this study elucidates the hormonal dynamics and gene regulatory networks underlying axillary bud germination in sugarcane, providing candidate gene resources for breeding high-yield varieties with enhanced emergence and tillering capacity. Thank you for pointingthis out. We agree with this comment. Therefore, We have shortened and streamlined the Abstract to make it more concise. Specifically, we reduced detailed method descriptions, focused on the core findings (hormonal dynamics, key modules, and hub transcription factors), and improved the logical flow of the summary. The revised Abstract is now more accessible and highlights only the most essential results. . Mention exactly where in the revised manuscript this change can be found – lines:13-25.

“[updated text in the manuscript]”

Comments 2: Keyword terms are generally not duplicated in the title.

Response 2: Keywords: sugarcane; bud; transcriptome; hormone; tillering; transcription factor

Thank you for pointing this out. We have revised the keywords to avoid duplication with the title. The new keywords are: sugarcane; bud; transcriptome; hormone; tillering; transcription factor. This change is marked in red in the revised manuscript.

“[updated text in the manuscript]”

Comments 3:  Please check the text for typos and errors.
Response 3:  Thank you for this suggestion. We have carefully reviewed the entire manuscript and corrected any typographical and grammatical errors. All other errors identified have been corrected accordingly.

“[updated text in the manuscript]”