Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study systematically investigates the impact of exogenous ABA on pollen development and function in Arabidopsis thaliana. By integrating phenotypic observations, cytological analyses, and transcriptomic profiling, the authors reveal that ABA inhibits pollen germination and pollen tube elongation—leading to reduced silique length and seed set—through the reprogramming of mRNA and lncRNA expression networks. The research is comprehensive, the data are robust, and the analyses are insightful. However, several issues need to be addressed to improve clarity, depth, and scientific rigor before the manuscript is suitable for publication.

1. Please supplement the specific developmental stages of anthers at the time of ABA treatment on Arabidopsis inflorescences, as well as key histological sections of anther development after treatment. In the silique results provided by the authors, the significant reduction in seed number does not conclusively indicate whether the abnormality is caused by defects in anther or pollen development. Histological sections of anther development are necessary to demonstrate abnormalities in pollen development.
2. This study used only a single concentration (100 μmol/L) of ABA treatment and did not explore the dose-dependent effects of different ABA concentrations. Furthermore, although the transcriptomic changes had significantly diminished by 22 hours, observations over a longer time course might reveal delayed effects or recovery mechanisms of ABA. It is recommended that the authors provide a rationale for the chosen ABA concentration or supplement phenotypic data under treatments with different concentrations.
3. It is recommended to include a concise regulatory model diagram in the Discussion section to visually illustrate how ABA affects pollen function through the mRNA-lncRNA network, thereby enhancing the visual impact and dissemination of the article.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Review on the ms. plants-4169803

The manuscript addresses a very interesting problem and presents a very comprehensive analysis of the effects of exogenous ABA treatment on pollen function and anther transcriptome. In Arabidopsis thaliana, it integrates phenotypic, cytological and RNA-seq approaches. The strengths of the study are the richness of the data and the joint analysis of mRNA, transcription factor and lncRNA results, as well as the use of a network approach.

However, the work in its current form is largely correlative and requires methodological refinement, stronger statistical support and more cautious mechanistic interpretation at several points. Clarification of the bioinformatic analysis, explicit hypothesis formulation and a more integrated interpretation of the results require more thought.

 

Comments on ABA: 100 µM ABA is a relatively high concentration, and indeed ABA is known to inhibit pollen germination and pollen tube growth, especially in in vitro systems. Therefore, the choice of concentration is a critical point. To what extent is 100 µM considered physiologically relevant in anther tissue?

In the case of in vitro pollen germination, even 10–50 µM ABA can cause significant inhibition in many species. A concentration of 100 µM often falls into the “stress-mimetic” or strongly inhibitory range. In transcriptomic studies, it is more difficult to separate adaptive responses from acute stress responses at such a dose.

Therefore, I suggest that the authors discuss the physiological relevance of the 100 µM ABA concentration more critically, since in this range the direct germination inhibitory effect of ABA alone could explain the observed deterioration in pollen function.

Below my comments and suggestions:

 

Introduction

I suggest that the statements regarding the complexity of pollen formation be clarified at the beginning of the Introduction and that pollen be briefly highlighted as being particularly sensitive to subtle changes in hormonal balance and environmental stress, thereby establishing the relevance of the topic more strongly.

I suggest that when presenting the role of ABA, positive and negative regulatory effects are discussed more differentiatedly, especially considering that ABA serves as both a stress-adaptive and a reproductive inhibitory signal.

The role of lncRNAs should be presented more prominently in the Introduction, not only as additional regulatory elements, but as potential key players in the integration of hormonal stress response and reproductive regulation.

I suggest that the significance of transcriptomic approaches be justified from a methodological perspective, briefly highlighting why RNA-seq is suitable for mapping ABA-induced transient and dynamic gene expression responses.

I suggest supplementing the paragraph on hormonal and stress signaling networks with a reference to a literature that uses promoter motif profiling and transcription factor binding site analysis to investigate stress responses in Arabidopsis (e.g. Virág et al., 2025 https://www.mdpi.com/1422-0067/26/22/11152).

Authors should add a clear, testable hypothesis formulated at the end of the Introduction, as the manuscript currently states a general research goal but does not make it clear what specific mechanistic hypothesis the study aims to investigate.

 

Results

In Section 2.1, I suggest clarifying the experimental design and the number of biological replicates, as the current description does not make the extent of statistical robustness clear.

Can the effect of a female reproductive component be excluded, since shorter stamens and reduced number of seeds may not only indicate a pollen dysfunction?

In section 2.2. the authors should also quantitatively support the morphological observations, since currently DAPI and aniline blue staining are only descriptive.

 

2.3. In transcriptome studies:

Authors should justify the choice of time points, especially why the response peaks at 8 hours.

I suggest that they discuss in more detail the biological significance of the 22-hour rearrangement, as this may indicate an adaptive or compensatory response.

I suggest that the functional significance of the 10 common DEGs be analyzed in more detail, as these may be potential key regulators.

For all pairwise comparisons, the specific sample pairs must be clearly indicated in the figures, not just in the text (e.g. ABA_0.5 h vs UC_0 h, ABA_2 h vs UC_0 h, etc.), as this would significantly improve the readability of the figures.

I suggest that the figure captions be more detailed and self-explanatory, including the number of samples (n), the number of biological replicates, and the statistical test used.

The authors should indicate the exact p-values ​​or the definition of the significance levels used in each graph, not just by marking them with asterisks.

The color coding must be standardized throughout the manuscript (e.g., control is always the same color, ABA treatments are consistently shaded), because currently the visual consistency is not always clear.

From a bioinformatic perspective, FPKM is not an ideal choice for differential expression (DE) testing, especially in such time-series/multiple-timepoint experiments. The manuscript also shows that RNA-seq values ​​are plotted as FPKM (e.g., in the description of Figure 7F).

I suggest that the manuscript clearly separates (i) the count-based method used for DE testing and (ii) the normalized counts (TPM/normalized counts) used for visualization, because the use of FPKM for DE purposes is methodologically questionable and weakens the robustness of the conclusions.

Authors should highlight: FPKM is at most suitable for illustration, not for inference, because:

its normalization between samples does not handle compositional effects well (when many genes change, the ratios are distorted),

it is sensitive to side effects of gene length and library size correction,

counts fit the basic assumptions of DE models (negative binomial, voom), not FPKM.

if you want to report “normalized expression”, then: I suggest to use TPM (transcript-per-million) for visualization,

I suggest that qRT-PCR validation should not be tied to FPKM, but to the normalized quantities used by the DE pipeline (e.g. DESeq2 normalized counts / vst / rlog), because if DE is based on counts-model, then it is consistent if the presented RNA-seq values ​​also fit it.

Discussion:

I suggest that at the end of the Discussion, a coherent, integrated model be formulated that links:

ABA induction, transcription factor activation, metabolic reprogramming, lncRNA-mediated fine-tuning, inhibition of pollen function, Currently, these are rather juxtaposed observations.

The Discussion should clearly separate proven results from hypothetical conclusions, as they are partly blurred in the current text.

In this section, it is important that authors briefly reflect on methodological limitations (use of FPKM, time-point design, lack of validation), as this would increase the scientific credibility of the manuscript.

I suggest that the final paragraph be strengthened with specific, testable future directions, rather than just a general “further validation is needed” statement.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The topic of regulating the formation, germination, and growth of the male gametophyte is relevant from both practical and theoretical perspectives.
The methods are modern and objective.

However, some comments and questions arise.

Methods
1. Why was this relatively high concentration of ABA used?
2. Section 4.2 does not specify the wavelengths used for fluorescence microscopy.
3. Line 672 – which stereomicroscope was used?
4. The figures and often the text give the impression that ABA treatments were carried out for 0, 0.5, 2, 8, and 22 hours. The text should be carefully proofread.
Discussion
1. The discussion places a great deal of emphasis on stress. It is clear that ABA is a stress-inducing phytohormone, but your study did not involve stress.
2. The discussion heavily repeats the results.
3. There is a lack of discussion of whether this result depends on the concentration, time, type, and phase of treatment.
4. There is evidence that ABA stimulates pollen tube growth both in vitro and in vivo.
For example:
Zakharova, E. V., Khaliluev, M. R., & Kovaleva, L. V. (2022). Hormonal signaling in the progamic phase of fertilization in plants. Horticulturae, 8(5), 365.
Kovaleva, L. V., Voronkov, A. S., Zakharova, E. V., & Andreev, I. M. (2018). ABA and IAA control microsporogenesis in Petunia hybrida L. L. V. Kovaleva et al. Protoplasma, 255(3), 751-759.

... and others
5. According to your data, how is ABA treatment related to reactive oxygen species?

6. There are no clearly formulated conclusions.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my comments and answered all my questions satisfactorily; therefore, I accept the manuscript for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors for their detailed responses to my questions and comments, and for the revisions they made to the article. I was completely satisfied with the responses, and I have no new comments. I wish you continued success in your research.