Transcriptomic Insights into Salt Stress Tolerance Mechanisms in Melia azedarach: 24-Epibrassinolide-Mediated Modulation of Auxin and ABA Signaling Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

can be accepted after few minor corrections

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper "Transcriptomic Insights into Salt Stress Tolerance Mechanisms in Melia azedarach: 24-Epibrassinolide-Mediated Modulation of Auxin and ABA Signaling Pathways" examines the role of 24-epibrassinolide (EBR) in alleviating the effects of salt stress in Melia azedarach through transcriptomic analysis. The study offers significant insights into plant hormone signaling pathways under salt stress; however, it is hindered by methodological limitations, analytical deficiencies, and interpretative challenges that compromise the reliability and breadth of its conclusions.

Introduction

Although auxin and ABA are essential to the study's title and objectives, the Introduction presents salinity as a general issue while leaving out important background information. Two well-known regulators of salt stress responses are auxin and ABA. For instance, auxin-mediated root plasticity is essential for salt, and ABA rises in saline environments to trigger adaptive reactions like stomatal closure. Nevertheless, neither auxin nor ABA signaling are mentioned in the manuscript, nor is there any reference to research on hormone crosstalk with brassinosteroids. This omission creates a conceptual gap because brassinosteroids are known to interact with auxin and ABA pathways under stress. Similar to this, M. azedarach is claimed to be novel ("significant tree species"), but the justification for concentrating on this species is not explained (e.g. its natural salt tolerance, unique phylogeny, or prior studies). 

Additionally, hypotheses and expected results are not adequately stated in the Introduction (e.g., how exactly EBR is predicted to modulate auxin/ABA). Lastly, the research gap and added value are unclear because the authors mention their earlier work on EBR in M. azedarach (ref. [36]) without describing how the current transcriptomic study builds upon it. Overall, the reader's comprehension of the study's rationale is hampered by the Introduction's inadequate defense of the auxin/ABA focus and its understatement of pertinent hormone biology.

Materials and methods

The transcriptome analysis was performed at a single time point (35 days post-EBR spraying), thus not reflecting the dynamic alterations in gene expression during salt stress adaptation. Time-course analyses are critical for comprehending the temporal dynamics of stress responses and hormone signaling pathways.

It is also not clear if each biological replicate is made up of one plant or a group of the 10 seedlings that were mentioned. The treatment plan is not normal: salt stress is applied first, then EBR is "sprayed" 15 days later, and the harvest is 35 days after spraying. This delayed EBR treatment is not supported or cited, and it complicates the understanding of EBR effects (there is no separate control group with EBR alone). The concentration and application method of "4% salt" and "EBR 1 mg/L" lack contextualization (e.g., salt molarity, rationale for dosage), and there is no specification on how uniform exposure among seedlings was achieved.

There are missing or inconsistent important details in the RNA-seq pipeline. The de novo assembly claims to use Trinity, but there are no assembly statistics (like total unigenes or N50) given, even though best practices say that metrics should be used to measure assembly quality. It was FPKM that was used to measure expression, but DESeq2 (which uses raw counts) was used for differential analysis. This shows that there is some confusion in the methods. The DEG threshold is "P < 0.05 and |log2FC| > 1," but it's not clear if P means raw or adjusted p-values. When there are thousands of tests, it is common to correct for multiple testing (for example, by using FDR). Using just raw P<0.05 probably gives you an inflated DEG list, as shown by the very high DEG counts that were reported.The methodology fails to address any multiple-testing correction or adjusted p-values, representing a significant oversight in accordance with statistical standards. 

Only one EBR concentration (1 mg/L) was tested in the study; no explanation for this particular dosage or investigation of possible dose-dependent effects was provided. This method restricts our knowledge of the ideal EBR concentration for reducing salt stress.

Only the KEGG database is mentioned in the paper, ignoring other significant databases such as Gene Ontology (GO), InterPro, or species-specific databases that might offer more thorough functional annotations.

The normalization techniques used for RNA-seq data analysis, which are essential for precise gene expression quantification, particularly when comparing various treatment conditions, are not sufficiently covered in the paper.

Results

Large amounts of descriptive data are presented in the Results section, but crucial statistical and contextual information is left out. Although the transition to differential expression is unclear, the sequencing quality metrics (Q30 scores, mapping rates) are sufficient. Although tens of thousands of DEGs are reported, neither the statistical confidence of enrichment findings nor the number of DEGs that are up- or down-regulated (apart from figures) are mentioned in the text. Though this conclusion is ambiguous and unsupported, the Venn diagram analysis (Figure 2b) is interpreted to "highlight [the] prioritized importance" of specific comparisons. It is impossible to determine which KEGG pathway enrichment results are actually significant because neither enrichment p-values nor FDR corrections are given, despite the fact that the results are listed (e.g., "ribosome," "oxidative phosphorylation"). The text contains no specific data or statistical values to back up claims like "a significant impact of salt stress."

Only when using raw P-values would the reports of 11,747 DEGs for SA vs. CK and 8,019 for E1 vs. SA make sense. It would be expected that many false positives would result from using a simple P<0.05 cutoff for so many genes. These findings should be interpreted cautiously in the absence of explicit adjustments. 94 vs. 54 DEGs are mentioned for the two comparisons in the plant hormone signaling analysis, but it is not made clear why these genes are the focus. Auxin and ABA genes are listed in the heatmap/map (Figure 4), but the narrative only reiterates the direction of change that was observed (e.g., “AUX1… genes consistently showed down-regulation”). These DEGs only have log2(FC) values, which means there is no way to tell how different or important they are.

The phrase "consistent expression patterns" sums up the qRT-PCR validation, but it offers neither correlation values nor actual expression data. Although "providing evidence for the reliability of the transcriptome data" is stated in the results text, no quantitative comparison—such as a scatter plot or correlation coefficient—is displayed. To assess consistency, the qPCR figure's error bars or p-values would be required. Furthermore, the validation is limited because only eight genes were tested. Overall, it is challenging to evaluate the veracity of the stated findings because the Results section provides a lot of numbers but little statistical analysis or critical interpretation.

Moreover authors are advised to provide the images of plant phenotype which correlates to growth changes. Otherwise study only seems to be descriptive in nature.

Discussion

The discussion mostly restates the findings with evidence from the literature, but it usually exaggerates the findings and ignores their limitations. For instance, the authors cite [3] to support their claim that EBR mitigates growth inhibition by affecting stomatal conductance and membrane stability; however, the study only measured height and diameter and did not collect any stomatal or physiological data. Although ABA can cause stomatal closure in salinity, this mechanism was not examined in this study. Therefore, it is speculative to associate EBR treatment with stomatal behavior. Similarly, EBR "restores growth" by upregulating auxin signaling genes, according to the discussion. The data only show transcript levels; neither hormone measurements nor functional assays are provided, despite the fact that brassinosteroids can interact with auxin pathways. The lack of measurement of auxin levels or transport, as well as the lack of analysis of root architecture or other growth parameters (beyond height), are not addressed in the paper. 

Additionally, the writers ignore intricate patterns of expression. Though some ARFs were down and one was up under salt, they stress that not all trends are consistent and that the majority of auxin-related genes were downregulated by salt and upregulated by EBR. There is no discussion of this variation in the auxin/ABA gene responses. The ribosomal genes, for example, continued to be downregulated despite EBR (Figure 3d), which runs counter to the assertion of restored growth capacity but is not addressed. Prior research in other species is cited in the discussion as "reinforcing" these mechanisms, but M. azedarach differences are not critically compared. The study's limitations—that is, the fact that only one time point was looked at, there was no EBR-only control, and hormone levels were not validated—are not acknowledged.

Coordinated gene expression "collectively enhances" stress adaptation, according to the concluding statements, but this is essentially a summary rather than a critical interpretation. Figure 6 presents the speculative model without any supporting data. In its current form, the Discussion tends to ignore contradictory or ambiguous aspects of the findings and instead asserts causal relationship from correlative data.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

REVIEWER COMMENTS

Melia azedarach L., commonly known as Chinaberry or Persian lilac, is a fast-growing deciduous tree native to South and Southeast Asia. It has been widely introduced to tropical and subtropical regions for ornamental use, timber production, and traditional medicine, although its toxicity requires caution. The species exhibits notable adaptability to drought and poor soils, but its invasive potential in non-native ecosystems limits its suitability for reforestation without careful management.

In this study, the authors aimed to elucidate the molecular mechanisms underlying salt stress responses in Melia azedarach L. and to assess how 24-epibrassinolide (EBR) modulates these responses, using transcriptomic analysis to identify key genes and pathways involved in EBR-enhanced salt tolerance.

TITLE: No comment.

ABSTRACT: No comment.

KEYWORDS: The current keywords are suboptimal. I suggest the authors consider the following alternatives for improved discoverability and specificity: Melia azedarach L.; Salt stress (or "Soil salinity" / "NaCl stress"); 24-epibrassinolide (EBR) / Brassinosteroids; Transcriptomic profiling / RNA-seq; Differentially expressed genes (DEGs); Phytohormone signaling / Hormonal regulation; Salinity tolerance mechanisms.

INTRODUCTORY SECTION: The introduction is generally adequate. However, in line 41, the term “signal transmission” should be revised to “signal transduction” to reflect the appropriate biological context.

MATERIALS AND METHODS:

2.1. Plant Materials and Experimental Design: The current description lacks clarity and coherence. The authors are encouraged to consider the following revised version for improved readability and precision:

“The experiment was conducted in a greenhouse nursery at Sheyang Tourism Investment Development Co., Ltd. (Sheyang County, Jiangsu Province, China) using one-year-four-month-old potted seedlings of Melia azedarach from the Sheyang seed source, grown under uniform conditions as per previous methods [36]. A randomized block design was applied with 90 seedlings divided into three treatments (10 seedlings × 3 replicates): (i) control (CK), (ii) 4% salt stress (SA), and (iii) 4% salt stress + 1 mg/L 24-epibrassinolide (EBR) (E1). EBR was foliar-sprayed 15 days after salt treatment, and leaf samples were collected 35 days post-EBR application, immediately frozen in liquid nitrogen, and stored at −80°C for RNA extraction.”

Furthermore, throughout the manuscript, percentages should be consistently expressed using the “%” symbol. Please revise accordingly.

Transcriptomics Methodology: The methods section is generally well-structured and adheres to standard transcriptomic workflows. However, a few enhancements could improve reproducibility and transparency:

1. Specify the RNA integrity threshold used for library construction (e.g., RIN > 7).
2. Include transcriptome assembly metrics (e.g., N50, total contigs) and clarify the extent of functional annotation (e.g., inclusion of GO, NR databases).
3. Indicate whether multiple-testing correction (e.g., FDR adjustment) was applied when identifying DEGs.
4. For qPCR validation, provide details on primer efficiency (e.g., 90–110%) and confirmatory results from melting curve analyses.

While no critical methodological gaps were identified, incorporating these details will significantly strengthen the manuscript’s rigor.

RESULTS:
3.1. Growth Changes: The statement that “EBR application substantially alleviated these salt-induced interferences, indicating its protective role against salinity stress (Figure 1)” appears to be overstated. As shown in Figure 1a and 1b, while EBR exhibited some alleviating effects, the differences in growth parameters between EBR and SA treatments were not statistically significant. The authors are advised to present these findings more cautiously in the revised manuscript.

The remaining results sections should be carefully re-evaluated for completeness, logical flow, and language clarity.

DISCUSSION: The discussion, in its current form, lacks depth and does not adequately convey the scientific contribution of this study within the context of existing literature. The authors are encouraged to integrate and compare their findings with the following two relevant studies:

• Ebeid, A., Mahmoud, A., Gahory, A., & Soliman, Y. (2022). Response of Melia azedarach L. tree seedlings to the addition of salt water and yeast. SVU-International Journal of Agricultural Sciences, 4(2), 210-223. doi: 10.21608/svuijas.2022.133727.1207
• Zhang, J., Fang, H., Jiang, J. et al. Analysis of transcriptomic profiles and physiological traits of exogenous 24-epibrassinolide alleviating salt stress in Atractylodes macrocephala Koidz.. Plant Biotechnol Rep 18, 161–175 (2024). https://doi.org/10.1007/s11816-023-00874-1

A meaningful comparison with these studies will help to situate the authors’ findings within the broader scientific discourse and underscore the novel contributions of this work.

Additionally, a critical reflection on the strengths and limitations of the study—such as the absence of gene validation experiments—should be included. This will offer a more balanced perspective and establish a solid foundation for future research directions.

CONCLUSION: The conclusion section should be revised to reflect the changes incorporated in response to the reviewer’s comments. It should synthesize the key outcomes more cautiously and align closely with the revised results and discussion sections.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

See comments!

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have improved the MS as suggested and now it stands fit for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

No further comments.