Exogenous Paclobutrazol Promotes Tiller Initiation in Rice Seedlings by Enhancing Sucrose Translocation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for submitting your manuscript entitled “Exogenous paclobutrazol promotes tiller initiation in rice seedlings by enhancing sucrose translocation.” The topic is relevant and the study provides promising insights into how paclobutrazol and seeding rates affect tiller initiation, sucrose dynamics, and carbohydrate metabolism in rice seedlings.
However, the manuscript in its current form requires substantial revisions to improve clarity, scientific rigor, methodological transparency, and the strength of the conclusions.

Below you will find numbered comments that should be addressed in detail in your revised version.

1. The Introduction is overly long and contains repeated ideas.

Please streamline the background, remove redundancy, and clearly articulate the specific knowledge gap that this study seeks to address. Highlight more precisely what remains unknown about PBZ, sucrose transport, and low-position tiller initiation.

2. The novelty of the work is not sufficiently emphasized.

Previous studies have investigated PBZ effects on plant height, tillering, and carbohydrate metabolism. You should explicitly state what is new in your experimental approach or findings compared with existing literature.

3. Important methodological details are missing.

The Materials and Methods section requires additional information to ensure reproducibility. Please clarify:

• Environmental conditions during seedling cultivation (temperature, humidity, light).
• Whether trays were randomized or blocked.
• How spray volumes were standardized.
• Exact number of biological and technical replicates per measurement.
  These details are essential for methodological transparency.

4. Statistical analysis needs clearer description.

The manuscript currently lacks information about:

• The type of ANOVA used (two-way, repeated measures, fixed vs random factors).
• Post-hoc tests applied.
• Assumptions evaluation (normality, homogeneity).
• Exact p-value thresholds.
  Please include a dedicated paragraph describing the statistical framework in detail.

5. Several figures require redesign for improved clarity.

Figures are difficult to follow due to inconsistent label formats, small fonts, unclear axis titles, and overly compressed panel layouts. Please revise figures to meet high-quality publication standards and ensure that all axes include units.

6. Some conclusions overinterpret the results.

Mechanistic claims regarding the interaction among PBZ, cytokinin biosynthesis, strigolactones, and sucrose signaling are not fully supported by your measurements. Because SLs, auxin distribution, and bud meristem status were not analyzed, these statements should be softened or framed as hypotheses rather than confirmed mechanisms.

7. The link between sucrose accumulation and cytokinin activation should be presented more cautiously.

Although supported by literature, no direct gene-expression evidence for cytokinin biosynthesis pathways is provided. Please avoid implying causality beyond what your data demonstrate.

8. Enzymatic activity interpretation requires moderation.

Inferring enhanced TCA cycle activity from ICDHc alone is insufficient. Please revise the discussion to avoid overstating physiological conclusions that are not directly supported by the data.

9. The gene expression analysis lacks important validation details.

Please include:

• Primer efficiency measurements.
• Melt curve analyses.
• Distinction between biological and technical replicates.
• Reference gene validation for stability.
  This information is necessary to support reliable qRT-PCR interpretation.

10. The Discussion contains large sections that repeat the Results.

Please reduce descriptive repetition and focus on discussing the biological significance of the findings. Integrate the discussion more concisely with current literature.

11. Several figure and table captions need fuller explanations.

Captions should allow readers to understand each figure independently. Please add:

• Clear definitions of treatments.
• Units.
• Number of replicates (n).
• Statistical tests used.
• Whether bars represent SD or SE.

12. English language requires improvement throughout the manuscript.

There are frequent issues with grammar, sentence structure, and cohesion. A thorough English revision—preferably by a professional language editor—is recommended to ensure clarity and readability.

13. References are appropriate but could be expanded.

Please consider citing additional recent studies related to sucrose signaling, PBZ mode of action, and tiller physiology to strengthen the scientific context.

14. The Conclusion should be more concise and avoid overgeneralization.

Currently, the conclusion reiterates mechanistic claims that exceed the scope of the collected data. Please limit conclusions to insights directly supported by your results.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear All,

The manuscript explores the physiological mechanisms by which foliar application of paclobutrazol (PBZ), combined with varying seeding rates, enhances early tiller initiation in hybrid rice seedlings. The authors assess sucrose transport, hormonal regulation, and associated enzymatic activities under controlled tray-seedling conditions. The experimental structure is sound, and the findings are relevant for optimizing rice mechanized transplanting systems.

However, while the study is well executed and contributes novel insights, the manuscript has critical methodological gaps, particularly concerning the description of the substrate used for seedling trays, which is central to interpreting the physiological responses observed. 

* Strong Points *

• The study integrates multiple levels of analysis: morphological (tiller counts, seedling vigor), biochemical (soluble sugars, starch), hormonal (cytokinins, gibberellins), and molecular (qRT-PCR of OsSUT1 and OsSUT4).

• It clearly contrasts low vs. high seeding rates under PBZ, GA₃, and control conditions, supported by appropriate statistical treatment.

• Enzyme activities (PFK, S-AI, ICDHc, PK) related to sugar catabolism and energy provision for tillering are appropriately measured and linked to phenotype.

• The graphical abstract (Figure 8) synthesizes the proposed physiological pathway effectively.

• The work has clear applied value for improving rice seedling quality in mechanized systems.

* Weaker Aspects and Major Revisions Needed *

1. Substrate Composition – Critical Missing Information

The manuscript states in Page 3, Line 97 that seedlings were grown in “standard 9-inch seedling trays,” but fails to describe:

• The physical and chemical composition of the substrate (soil, peat, vermiculite, etc.)

• Nutrient content, pH, CEC, or texture of the medium

• Whether fertilizers or base nutrients were added

• Watering and irrigation regime (e.g., misting, subirrigation)

• Substrate moisture retention capacity, which affects sucrose translocation, enzyme activity, and hormone dynamics

Reviewer note: Without this information, the reproducibility of the results and interpretation of nutrient-dependent metabolic changes is compromised. It also limits the external validity of the study for agronomic recommendations.

2. Methods – Additional Clarifications Required

• Page 3, Line 100–104: Although germination and tray transfer protocols are described, nothing is reported about the root-zone temperature, which influences tiller emergence and sugar metabolism.

• Page 4, Lines 115–120: Seedling quality parameters are well listed, but it should be stated whether destructive sampling was conducted per tray or if tray reuse occurred.

• Table 1 (Page 3): Useful treatment design, but the authors should clarify whether the tray location in the field was randomized or fixed (potential position effects).

* Language and Style *

The manuscript is clearly written and well-organized. Minor stylistic polishing would improve scientific tone in some cases:

• Page 1, Line 12: "Paclobutrazol (PBZ) is a plant growth regulator..."
  → Suggest: "Paclobutrazol (PBZ) is a triazole-class growth retardant widely applied to modulate tillering and stem elongation in rice."

• Page 6, Line 260: "enhance chlorophyll content in leaves."
  → Suggest: "increase foliar chlorophyll concentration, improving photosynthetic capacity."

Up to 15 such refinements could enhance precision and flow.

* Suggested Specific Fixes *

Page	Line	Issue	Suggested Fix
3	97	Missing substrate details	Describe substrate composition, pH, and nutrient availabilities.
3	100	Germination under high RH	Specify light/dark and humidity control
4	113	Watering not described	Include irrigation regime
5	144	Soluble sugars	Indicate if glucose, sucrose, and fructose are distinguished
6	198	“Tillers were nearly absent”	Quantify: e.g., mean ± SD or n (%)
13	441	“Control seedling height”	Suggest: “Restrict shoot elongation and promote early tillering”

* Experimental Rigor *

The physiological measurements (sugar, hormones, enzyme activity) are sound and the sample sizes (n = 3 per treatment) are adequate for preliminary studies. However, greater attention to substrate standardization and environmental conditions (e.g., light intensity, humidity) would increase robustness.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

So, first off, the topic is relevant and the experiment on seeding density × PBZ effects in rice is interesting. You can tell the authors put real effort into the setup. The manuscript reads fairly well, and I could follow the logic without getting lost. The combination of physiological, biochemical and gene expression data adds value — it’s not just descriptive. The conclusion (PBZ boosting early tillering, probably via sucrose metabolism, hormone changes, chlorophyll, etc.) feels reasonable based on what’s shown. The figures are fine; I had no trouble reading them.

However, a few things need tightening before this is publishable.

The statistics section needs more explanation — this is my main concern. It isn’t clear whether assumptions were checked, or how the factors were treated in the model (fixed? random? mixed?). Also, the post-hoc test isn’t named. With multiple factors involved, these missing details matter, otherwise readers don’t know how robust the comparisons actually are.

The introduction is OK, but it gets repetitive in places, especially when discussing seeding density and transplanting limitations. It could be shortened a bit — right now it feels like you circle the same ideas twice.

Since you already included screening data in the appendix, it would be helpful to briefly explain why you chose the final PBZ concentration. If possible, compare it to what’s actually used in fields or in seedling nurseries. And maybe mention potential drawbacks like delayed panicle development or excessive dwarfing — just to balance the interpretation a bit.

For reproducibility: the manuscript would benefit from more detail about growth conditions (light intensity, temperature, substrate or soil type, etc.), because these variables can strongly affect rice metabolism and tillering. Also I myself have never done GA analysis with ELISA, the authors cite reference and kits for method however I would ask have they done any quality controls previously that would verify ELISA method performance for GA-s? GA-s are present in plant marices in very low concentrations, even in the case of seedlings even with, with exception of growing plant parts usually. As an LC-MS/MS expert I'm always a bit skeptical about ELISA in general (not as selective and linearity can be problematic). Can the authors provide ELISA performance and results maybe as a supplement so I can check? Otherwise on figure E F G H paralells exhibit very low standard deviation for n=3. How much plant material was used for determining the GA contents in your case? Can you provide amufacturer instructions for reviewing?

One more scientific comment — the sucrose transporter gene expression result is interesting but feels a bit isolated. The discussion could connect it briefly to broader sugar signaling frameworks (T6P, SnRK1). Doesn’t need to be long — even one sentence would make the result feel better integrated.

Lastly, since the work uses only one genotype in controlled conditions, I’d suggest adding a short limitations note. It sets boundaries on applicability, and editors/reviewers usually expect that.

Overall, the paper is good and just needs clarifications and some polishing. 

Comments on the Quality of English Language

The English language is generally understandable, and the manuscript is written in a scientific style appropriate for an agronomy journal. However, the text contains numerous grammatical inconsistencies, overly long sentences, repetition of ideas, and occasional awkward phrasing that affects clarity and flow. Technical terminology is used correctly, but sentence structure and punctuation require improvement for better readability. Several sections—particularly the Introduction and Discussion—would benefit from professional English editing to improve conciseness, remove redundancy, and ensure consistent tense, article use, and sentence construction.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf