Comparative Transcriptome Analysis Reveals Molecular Indicators of Embryogenic Initiation Divergence Between Rice Varieties CXJ and 9311 During Microspore Culture

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this work, the authors compared japonica (CXJ) and indica (9311) rice during isolated microspore culture using time-course RNA-seq (0, 5, 10 dpi). They integrated viability and callus induction phenotypes with transcriptomic clustering, GO/KEGG enrichment, and transcription factor family analysis. They found CXJ maintains stable developmental and hormone-regulated programs, while 9311 shifts into stress-response and maladaptive metabolic pathways, explaining divergent embryogenic competence. This work reports good results and is well written. I have a few comments and suggestions.

Please report the precise developmental stage (e.g., late uninucleate) with cytology (DAPI/acetocarmine) and staging QC.

The composition of isolation/culture media (basal salts, carbon source, hormones/osmolytes, casein hydrolysate, pH, gelling agent) is not provided; without it, the phenotype (especially 9311 lethality) is not reproducible.

A fixed 12-day 4 °C pretreatment is used for both genotypes; justify with data or genotype-specific optimization, as pretreatment duration strongly affects androgenesis.

RNA-seq replicate number per genotype × time is unclear; state n and show replicate-level QC.

Mapping both CXJ and 9311 reads to a single reference can inflate apparent DE in the indica line. Have authors considered using SNP-aware mapping?

You compute FPKM/TPM but then call DE with DESeq2, which requires raw counts and its own size-factor normalization. Please make it clear.

Please expand the discussion and discuss the obtained results for japonica and indica rice genotypes with comparison to previous literature.

Author Response

Comments 1: Please report the precise developmental stage (e.g., late uninucleate) with cytology (DAPI/acetocarmine) and staging QC.

Response 1: Thank you for this suggestion. We have included this detailed description in the Methods section (Section 2.1, Lines 118-125) of the revised manuscript. "A slightly modified 4′,6-diamidino-2-phenylindole (DAPI) staining method as previously described in our prior work [10] was used to determine the developmental stage of microspores. Briefly, microspores were fixed in Carnoy’s Fluid (ethanol:acetic acid = 3:1) for 10 minutes. After removing the supernatant, 10 µL of double-distilled water and 10 µL of 0.1 mg/mL DAPI staining solution were added, mixed thoroughly, and incubated at 25°C in the dark for 10 minutes. The samples were then observed under a fluorescence microscope. Panicles containing microspores at the late uninucleate stage were selected for subsequent experiments."

Comments 2: The composition of isolation/culture media (basal salts, carbon source, hormones/osmolytes, casein hydrolysate, pH, gelling agent) is not provided; without it, the phenotype (especially 9311 lethality) is not reproducible.

Response 2: Thank you for pointing this out. This detailed composition has been provided in Section 2.3 (Lines 141-147) of the revised manuscript to ensure reproducibility. "The isolation and culture medium consisted of NB basic medium (comprising N6 macronutrients and B5 micronutrients and vitamins), supplemented with 75 g/L maltose as the carbon source, 0.75 g/L glutamine, 2 mg/L 2,4-D, 0.5 mg/L KT (Kinetin), and 0.976 g/L MES (2-(N-morpholino)ethanesulfonic acid). The pH was adjusted to 5.8. As this is a liquid culture system, no gelling agent was used. The medium was filter-sterilized using a 0.22 μm PES (Polyethersulfone) membrane (Merck Millipore, Darmstadt, Germany)."

Comments 3: A fixed 12-day 4 °C pretreatment is used for both genotypes; justify with data or genotype-specific optimization, as pretreatment duration strongly affects androgenesis.

Response 3: We agree that the duration of cold pretreatment is critical and genotype-dependent. Our laboratory’s previous research [8] has shown that a 10-day cold treatment is necessary for the initiation of microspore embryogenesis. Based on extensive empirical experience, a 12-day pretreatment at 4°C was adopted for both genotypes in this study to ensure consistent induction of embryogenic competence. We acknowledge that optimal pretreatment conditions for indica rice remain underexplored and agree that further investigation into genotype-specific pretreatment durations is warranted. We have included this rationale in Section 2.2 (Lines 128-131). "In accordance with established protocols derived from previous studies [8], which demonstrated that a 10-day cold treatment is required to initiate microspore embryogenesis, a 12-day pretreatment at 4°C was applied to both genotypes in this study to ensure reliable induction of embryogenic competence."

Comments 4: RNA-seq replicate number per genotype × time is unclear; state n and show replicate-level QC.

Response 4: Thank you for pointing this out. We have stated the replicate number in Section 3.2 (Lines 241-242). Replicate-level QC metrics (e.g., Q30, GC%) were provided in Table S1 and was referenced in the main text. "RNA sequencing was performed on microspores of both cultivars harvested at 0, 5, and 10 dpi, and each sample group (genotype × time point) included three biological replicates (Table S1)."

Comments 5: Mapping both CXJ and 9311 reads to a single reference can inflate apparent DE in the indica line. Have authors considered using SNP-aware mapping?

Response 5: This is a valid concern. We aligned all reads to the japonica Nipponbare reference genome, which may introduce mapping bias against the indica genotype. Although we did not use SNP-aware mapping in this study, the overall mapping rates were high and comparable between genotypes (Table S1). To minimize the impact of genotypic variation, we focused on differentially expressed genes (DEGs) identified through pairwise comparisons between genotypes at the same time point. Moreover, true DEGs affecting microspore culture efficiency (rather than inherent genotypic differences) were further prioritized by Venn diagram analysis to identify culture-responsive genes specific to each genotype. We acknowledge this limitation and will pay attention to this detail in future research.

Comments 6: You compute FPKM/TPM but then call DE with DESeq2, which requires raw counts and its own size-factor normalization. Please make it clear.

Response 6: Thank you for highlighting this. We have emphasized this distinction in Section 2.8 (Lines 185-189) to avoid confusion. "The expression levels of genes were quantified as FPKM (Fragments Per Kilobase per Million mapped reads) values for visualization and comparative purposes. However, for the identification of differentially expressed genes (DEGs), the raw read counts were used as input for the DESeq2 package (version 1.38.3) with significance thresholds of |log2FoldChange| > 1 and P-value < 0.05."

Comments 7: Please expand the discussion and discuss the obtained results for japonica and indica rice genotypes with comparison to previous literature.

Response 7: We thank the Reviewer for this suggestion. We have now expanded the Discussion section to explicitly contextualize our findings within the existing literature on the well-documented genotypic disparity between japonica and indica rice. We have added a sentence in the opening paragraph of the Discussion (Lines 421-424). "This genotypic disparity is well-recognized, with indica rice consistently exhibiting significantly lower microspore culture efficiency and being more recalcitrant to in vitro culture compared to japonica subspecies, despite incremental improvements in haploid yield [5]."

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well organized and clearly written. The information derived from results is potentially of interest and could represent important impact on biotechnological use of recalcitrant to in vitro culture rice varieties. However, results are presented in a very general way, with no highlight of gene(s) that potentially could represent biomarkers for successful callus induction from microspores derived from the CXJ variety. Since there is no further exploration on DEGs of interest at experimental level, the availability of supplementary material derived from transcriptome analyses and supporting the Figures is crucial but lacking in the current manuscript version. Moreover, the title claims the identification of "molecular determinants for embryogenic competence" but no gene ID or name from any group of differentially expressed genes is mentioned in the manuscript.

Specific comments:

Materials and methods

Lines 105-107. Are there differences between the timing (days after sowing or germination) of panicle collection between the japonica and indica rice varieties used in the study? 

Line 110. Please point out the low temperature used in the study.

Results

Line 188 and Figure 1a. It would be helpful to point by arrow some callus forming microspores in CXJ, Figure 1a at day 10, as well as some dead microspores in both varieties at day 5 to discern dead vs live microspores.

Lines 300-318. The paragraphs are in reference to both varieties in similar way, or there are differences for the described GOs and KEGG pathways between varieties. Please clarify. I could not understand from Figure 5 a and b whether the pathways are more enriched in DEGs from CXJ or 9311.

Figure 6 is difficult to understand. There is no Supplementary information in support to this Figure. Since most TF families are represented by many members, a supplementary table indicating the log2FC and adj. p-value with geneID, gene name and description would clarify which members are up-regulated, not-changed or down-regulated for each variety at the represented time points of 5 and 10 days upon induction. Such information is crucial to understand gene expression regulation differences between varieties.

Normalized counts and log2FC with p-values, adj. p-values for DEGs should be made available for research articles dealing with transcriptomes. Genes belonging to each group of K-means clustering are also usually available as supplementary table.

 

Author Response

Comments 1: Lines 105-107. Are there differences between the timing (days after sowing or germination) of panicle collection between the japonica and indica rice varieties used in the study?

Response 1: Thank you for raising this point. Based on the available varietal information and actual cultivation practices in our experimental field, there was no significant difference in the panicle collection time (days after sowing) between the two rice varieties used in this study. Both were sown at the same time and managed under identical conditions. Panicles were collected based on morphological criteria (booting stage with swollen flag leaf sheath) rather than days after sowing, which is standard practice to ensure developmental synchrony. We will clarify this in the revised manuscript (Section 2.1 Lines 113-114). "There was no significant difference in the panicle collection time between the two rice varieties used in the study."

Comments 2: Please point out the low temperature used in the study.

Response 2: Thank you for this suggestion. The low-temperature pre-treatment was conducted at 4 °C for 12 days. We added this in Section 2.2 (Lines 128-131). "In accordance with established protocols derived from previous studies [8], which demonstrated that a 10-day cold treatment is required to initiate microspore embryogenesis, a 12-day pretreatment at 4°C was applied to both genotypes in this study to ensure reliable induction of embryogenic competence."

Comments 3: Line 188 and Figure 1a. It would be helpful to point by arrow some callus forming microspores in CXJ, Figure 1a at day 10, as well as some dead microspores in both varieties at day 5 to discern dead vs live microspores.

Response 3: We thank for this suggestion. We have now revised Figure 1a, and the figure legend has been updated accordingly to describe these annotations (Lines 233-236). "Arrows in the images point to representative examples of the four distinct types of cells/structures observed: Undeveloped microspore (UM), Multicellular structure (MS), Callus (C), and Apoptotic cell (AC). Two representative examples are indicated for each type." Also, section 3.1 (Lines 211-218) was also updated. "In this system, several distinct cellular states were observed: Undeveloped microspore (UM) refers to microspores that either remained at the initial state or had survived by 5 or 10 dpi without further development; these are spherical with visible germ pores. Multicellular structure (MS) denotes cell clusters that had successfully initiated embryogenic divisions by 5 dpi, appearing as opaque black structures similar in size or slightly larger than UMs. Callus (C) developed from MS by 10 dpi, forming aggregated clumps. Apoptotic cell (AC) represents microspores that underwent cell death during culture, exhibiting plasmolysis and a notably smaller size compared to living microspores."

Comments 4: Lines 300-318. The paragraphs are in reference to both varieties in similar way, or there are differences for the described GOs and KEGG pathways between varieties. Please clarify. I could not understand from Figure 5 a and b whether the pathways are more enriched in DEGs from CXJ or 9311.

Response 4: Thank you for this suggestion. We have revised the text in Section 3.5 (Lines 354-391) to make it clearer. "

Functional enrichment analysis of DEGs between CXJ and 9311 revealed dynamic, stage-specific adaptations that underscore the divergent strategies employed by the two varieties (Figure 5). For all subsequent descriptions, 'up-regulated' refers to higher expression in CXJ relative to 9311 at the given time point, and 'down-regulated' refers to lower expression in CXJ.

At 5 dpi, cellular components were predominantly localized to membrane systems, with significant enrichment in plasma membrane (768 up- vs. 535 down-regulated), cell periphery (938 up- vs. 650 down), and membrane (1391 up- vs. 964 down), indicating substantial membrane reorganization. Molecular functions centered on catalytic activities (2216 up- vs. 1607 down), though cytoskeletal motor activity showed more downregulation (8 up- vs. 40 down). Kinase activity was also enriched (434 up- vs. 387 down). Biological processes involved stress adaptation, with response to stress (867 up- vs. 625 down) and response to stimulus (1369 up- vs. 982 down) showing strong enrichment, alongside carbohydrate metabolic process (229 up- vs. 192 down).

KEGG pathway analysis at 5 dpi confirmed this stress-prone state. Protein processing in endoplasmic reticulum (83 up- vs. 27 down), plant-pathogen interaction (79 up- vs. 27 down), and α-linolenic acid metabolism (23 up- vs. 8 down) all showed marked enrichment. Starch and sucrose metabolism was also enriched but with more balanced regulation (41 up- vs. 45 down). Additional pathways including biosynthesis of other secondary metabolites (8 up- vs. 3 down) and galactose metabolism (22 up- vs. 12 down) further demonstrated metabolic dysregulation of 9311.

By 10 dpi, membrane localization persisted with plasma membrane (822 up- vs. 657 down), cell periphery (978 up- vs. 812 down), and membrane (1575 up- vs. 1146 down) maintaining enrichment. Molecular functions shifted toward signaling mechanisms, showing enrichment in signal transduction (373 up- vs. 326 down), regulation of cellular process (373 up- vs. 326 down), and cellular response to stimulus (373 up- vs. 326 down). Enzymatic regulation was evident through hydrolase activity (746 up- vs. 664 down), kinase activity (470 up- vs. 466 down), and transferase activity (905 up- vs. 825 down).

KEGG analysis at 10 dpi revealed plant-pathogen interaction (90 up- vs. 36 down) and glutathione metabolism (50 up- vs. 16 down) as top pathways with strong enrichment. Starch and sucrose metabolism continued to be enriched (38 up- vs. 59 down), now showing a downregulation trend. Plant hormone signal transduction was significantly enriched (89 up- vs. 46 down), alongside brassinosteroid biosynthesis (4 up- vs. 8 down) and nitrogen metabolism (16 up- vs. 7 down).

This comprehensive analysis demonstrates distinct genotypic responses across time points, with 9311 showing persistent activation of stress-response pathways. Complete statistical details for all enriched terms, including gene ratios, p-values, and full gene lists, are available in Supplementary Table S4."

Comments 5: Figure 6 is difficult to understand. There is no Supplementary information in support to this Figure. Since most TF families are represented by many members, a supplementary table indicating the log2FC and adj. p-value with geneID, gene name and description would clarify which members are up-regulated, not-changed or down-regulated for each variety at the represented time points of 5 and 10 days upon induction. Such information is crucial to understand gene expression regulation differences between varieties.

Response 5: We sincerely thank the you for the critical suggestion. To address the request for detailed TF information supporting Figure 6: We have created Supplementary Table S6 ("Differentially expressed transcription factors (TFs) between CXJ and 9311 at 5 and 10 dpi"), which provides the complete list of differentially expressed TFs and their details.

Comments 6: Normalized counts and log2FC with p-values, adj. p-values for DEGs should be made available for research articles dealing with transcriptomes. Genes belonging to each group of K-means clustering are also usually available as supplementary table.

Response 6: To address the request for full DEG statistics and K-means gene lists: We have created two additional tables: Supplementary Table S2 ("Comprehensive list and annotation of all differentially expressed genes (DEGs) across all pairwise comparisons"): This master table contains the normalized counts, log2FoldChange, p-values for all significant DEGs in the key comparisons, along with comprehensive functional annotations. Supplementary Table S3 ("Gene membership and Z-score normalized expression values for K-means clusters"): This table lists all genes assigned to each K-means cluster (C1-C9), allowing readers to access the exact gene sets underlying the expression trends shown in Figure 3 and 4.

Reviewer 3 Report

Comments and Suggestions for Authors

The presented research entitled 'Comparative transcriptome analysis reveals molecular determinants of embryogenic competence divergence between rice varieties CXJ and 9311 during microspore culture' is interesting, timely and advancing. However, I have major concern with the terminology used. Consequently, I also do not agree to the title and recommend modification.

My concern is with the usage of the term 'determinants of competence', since the research does not explore the processes underlying the first moments underlying induction, but studies the initiation process, which is the realization of competence. Maybe the authors could agree to modify the title to ....molecular indicators of embryogenic competence initiation.... Furthermore, the terms of competence, induction and initiation must be introduced (Introduction part). Also, the relevance for predicting determinants of competence (genetic competence for being able to be induced + induction of competence) needs to be discussed versus the information coming from the initiation process (there might be many ways of initiation depending also on individuals!).

Without these improvements, the interpretation of the results, as it is written, is misleading and might influence other scientists into a wrong direction of theoretical thinking and interpretation, means avoiding further meaningful progress.

However, with the proposed corrections, this research is of high interest.

Hope, the authors feel that this is helpful.....

Author Response

Comment: The presented research entitled 'Comparative transcriptome analysis reveals molecular determinants of embryogenic competence divergence between rice varieties CXJ and 9311 during microspore culture' is interesting, timely and advancing. However, I have major concern with the terminology used. Consequently, I also do not agree to the title and recommend modification.

My concern is with the usage of the term 'determinants of competence', since the research does not explore the processes underlying the first moments underlying induction, but studies the initiation process, which is the realization of competence. Maybe the authors could agree to modify the title to ....molecular indicators of embryogenic competence initiation.... Furthermore, the terms of competence, induction and initiation must be introduced (Introduction part). Also, the relevance for predicting determinants of competence (genetic competence for being able to be induced + induction of competence) needs to be discussed versus the information coming from the initiation process (there might be many ways of initiation depending also on individuals!).

Response: We sincerely thank the Reviewer for their profound and insightful comment regarding the precise use of terminology, which has greatly improved the conceptual rigor and accuracy of our manuscript. We fully agree with the distinction between 'competence', 'induction', and 'initiation' and appreciate the guidance in aligning our claims more precisely with our data.

In direct response to the Reviewer's recommendation, we have implemented the following key changes:

Title Change: We have changed the title of the manuscript from "...molecular determinants of embryogenic competence divergence..." to "Comparative transcriptome analysis reveals molecular indicators of embryogenic initiation divergence between rice varieties CXJ and 9311 during microspore culture." We believe this new title accurately reflects that our study captures the molecular events during the realization (initiation) of embryogenic potential, rather than its initial acquisition.

Terminology Definitions: We have added a new paragraph in the Introduction (Section 1) to explicitly define the terms embryogenic competence, induction, and initiation within the context of microspore embryogenesis, citing appropriate literature (Khanday et al., 2019; Shi et al., 2025). This provides the essential conceptual framework requested (Lines 61-67). "In microspore embryogenesis, a clear distinction is made between several key phases. The process begins with the acquisition of embryogenic competence, which represents the latent potential of a microspore to switch its developmental fate [6]. This is frequently achieved through an induction process, most commonly triggered by stress pre-treatments which confer this competence, although alternative pathways exist [7]. Finally, the initiation phase constitutes the realization of this acquired competence, marked by active cell division and morphological changes that lead to callus formation."

Reframed Discussion: We have carefully reframed the Discussion to consistently interpret our results within the context of the initiation phase. Furthermore, we have expanded the discussion to include the requested comparison to previous literature, citing Tripathy (2021) [5] to highlight the well-documented genotypic disparity our study seeks to explain (Lines 420-424). "This study dissects the molecular basis underlying the stark contrast in microspore embryogenic initiation between japonica CXJ and indica 9311. This genotypic disparity is well-recognized, with indica rice consistently exhibiting significantly lower microspore culture efficiency and being more recalcitrant to in vitro culture compared to japonica subspecies, despite incremental improvements in haploid yield [5]."

We believe these revisions have successfully addressed the Reviewer's concern that the interpretation might be "misleading." Our findings now clearly position the identified molecular features as indicators characterizing the divergent initiation process, which serves as a diagnostic signature for the underlying difference in embryogenic competence. We again thank the Reviewer for this invaluable suggestion, which has significantly strengthened the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

This study conducts a comparative transcriptomic analysis between japonica and indica rice varieties to understand the differences in embryogenic competition during microspore culture, subjecting them to stress and analyzing the development of microspores on different days (0, 5, and 10). In addition, it identifies the differences between gene groups and their functions to explain why CXJ generates callus while 9311 leads to apoptosis.

The manuscript is interesting because of the subject matter it deals with, as well as the fact that rice is commercially important and the development of improved varieties is crucial to its development. However, some adjustments and clarifications should be made to improve the manuscript, especially in the materials and methods section, to be clear about the process that was developed. The introduction is fine, the results are adequate and show the analyses performed, and the discussion is interesting, but the final part should also be reinforced to highlight the importance of the work, as no conclusion is reached.

In addition, the following observations are made:

Specify the importance of collecting leaves between 9:00 and 10:00 a.m. (line 107).

Specify how the panicles were sterilized, as residues may remain depending on the solution used (line 115).

The explanation given at the end of the discussion is interesting; however, they do not discuss the relevance of these results, how they could be used in the future, what implications they have for obtaining cultivars, etc.

In addition, it would be advisable to provide a specific conclusion based on the results and the hypothesis or objective initially proposed.

It would also be advisable to consider continuing the experiment in the future to test whether the callus formed is viable or not for the CXJ variety.

In materials and methods, it is necessary to mention whether the culture media were homogeneous, what the culture media were made of, or what was used as a basis for choosing such media, as this may also affect the viability of the microspores.

Mention in the materials and methods section which genes were used to determine their gene expression and which are the basis for the subsequent principal component analysis and others, and also state what criteria were used to choose these genes.

Author Response

Comments 1: Specify the importance of collecting leaves between 9:00 and 10:00 a.m. (line 107).

Response 1: Thank you for raising this point. We have added the rationale to Section 2.1 (Lines 114-118) of the manuscript. "At the booting stage, identified by swelling of the flag leaf sheath, panicles were collected between 9:00 and 10:00 a.m. to avoid the high temperatures and intense sunlight of midday, which can induce heat stress and alter microspore physiology, thereby ensuring developmental consistency and high initial viability. "

Comments 2: Specify how the panicles were sterilized, as residues may remain depending on the solution used (line 115).

Response 2: Thank you for pointing this out. We have now added the detailed sterilization protocol to Section 2.3 (Lines 135-139). "After the cold pre-treatment, rice panicles were subjected to surface sterilization by immersion in a 10% (v/v) sodium hypochlorite (NaOCl) solution for 10 minutes. This was followed by five thorough rinses with sterile distilled water to completely eliminate any residual sterilant that could be toxic to the microspores.

Comments 3: The explanation given at the end of the discussion is interesting; however, they do not discuss the relevance of these results, how they could be used in the future, what implications they have for obtaining cultivars, etc.

In addition, it would be advisable to provide a specific conclusion based on the results and the hypothesis or objective initially proposed.

It would also be advisable to consider continuing the experiment in the future to test whether the callus formed is viable or not for the CXJ variety.

Response 3: Thank you for this essential suggestion. We have now added a standalone Future Perspectives section (Section 4.5, Lines 499-505) and a Conclusions section (Section 5, Lines 506-516) that addresses these points directly. "4.5 Future Perspectives: While this study focused on the molecular events during the critical initiation phase, a key future direction will be to test the regeneration potential of CXJ-derived calli into fertile plants, thus fully validating their embryogenic competence. Furthermore, functional validation of the candidate genes and TFs identified here, through overexpression or gene editing in 9311, will be crucial to confirm their roles as master regulators of embryogenic initiation."; "5 Conclusions: Our comparative transcriptome analysis demonstrates that the divergent embryogenic initiation outcomes between japonica CXJ and indica 9311 arise from starkly contrasting transcriptional programs during early culture. CXJ succeeds by maintaining transcriptional stability and activating developmental processes (e.g., hormone signaling, DNA replication) alongside resilience-conferring transcription factors (NAC, ERF, HSF). In contrast, 9311 fails due to a maladaptive overdrive of stress-response pathways (e.g., plant-pathogen interaction, ER stress) and futile metabolism, culminating in cell death. These findings provide both a diagnostic toolkit for assessing microspore potential and actionable targets, such as key TFs or stress pathways, for biotechnological strategies to overcome genotype-dependent barriers in rice doubled haploid breeding."

Comments 4: In materials and methods, it is necessary to mention whether the culture media were homogeneous, what the culture media were made of, or what was used as a basis for choosing such media, as this may also affect the viability of the microspores.

Response 4: We agree and have now provided the full detailed composition of the culture medium in Section 2.3 （Lines 141-147). "The isolation and culture medium consisted of NB basic medium (comprising N6 macronutrients and B5 micronutrients and vitamins), supplemented with 75 g/L maltose as the carbon source, 0.75 g/L glutamine, 2 mg/L 2,4-D, 0.5 mg/L KT (Kinetin), and 0.976 g/L MES (2-(N-morpholino)ethanesulfonic acid). The pH was adjusted to 5.8. As this is a liquid culture system, no gelling agent was used. The medium was filter-sterilized using a 0.22 μm PES (Polyethersulfone) membrane (Merck Millipore, Darmstadt, Germany)."

Comments 5: Mention in the materials and methods section which genes were used to determine their gene expression and which are the basis for the subsequent principal component analysis and others, and also state what criteria were used to choose these genes.

Response 5: We thank the Reviewer for prompting this clarification. We have added a statement in Section 2.7 to clarify that the transcriptome analysis was performed on a genome-wide scale. This means that all genes present in the reference genome annotation were included in the initial quantification and all subsequent analyses (e.g., PCA, differential expression). There was no pre-selection of genes; the criteria for identifying biologically relevant genes were applied after sequencing through statistical filters (e.g., adjusted p-value < 0.05 and |log2FoldChange| > 1 for DEGs). (Lines 176-178) "The analysis was performed on a genome-wide scale, encompassing all genes in the reference genome annotation. No pre-selection of genes was performed prior to sequencing."

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All concerns and recommendations have been adressed.

Author Response

Thank you for your positive feedback and confirmation that all issues have been resolved. We appreciate your time and insightful comments, which have greatly improved our manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors agreed to the proposed changes and revised crucial parts in the title and all over the text.

However, the according changes are not reflected in the Abstract (see first sentence ('embryogenic competence' is not corrected) and last sentence ('determinants' should be substitued) in the abstract plus in line 22 of the abstract: 'by 5 days, characterized by maladaptive induction of stress-response' - after 5d the authors should not refer to 'induction', that's wrong. These corrections must still be performed before publication.

 

Author Response

Comment: The according changes are not reflected in the Abstract (see first sentence ('embryogenic competence' is not corrected) and last sentence ('determinants' should be substitued) in the abstract plus in line 22 of the abstract: 'by 5 days, characterized by maladaptive induction of stress-response' - after 5d the authors should not refer to 'induction', that's wrong. These corrections must still be performed before publication.

Response: Thank you for this essential suggestion. As suggested, we have updated the abstract (Lines 15-31) to incorporate your feedback. "This study elucidates the key molecular features underlying the divergent embryogenic initiation divergence between japonica rice Chongxiangjing (CXJ) and indica rice 9311 during isolated microspore culture. Comparative transcriptome analysis across critical timepoints (0, 5, and 10 days post-culture initiation) revealed that while both varieties initially exhibit comparable microspore viability, CXJ maintains transcriptional stability and activates developmental programs (e.g., hormone signaling, DNA replication, cell morphogenesis), enabling sustained callus formation. In contrast, 9311 undergoes drastic transcriptome reorganization by 5 days, characterized by maladaptive activation of stress-response pathways (glutathione metabolism, MAPK signaling, ER stress) and futile metabolic reactivation (photosynthesis, starch degradation), culminating in near-total cell death and failed callus induction. Transcription factor dynamics further explain this divergence: CXJ specifically upregulates regulators coordinating development and stress resilience (NAC, ERF, HSF, GRAS, bZIP), while 9311 exhibits detrimental upregulation of FAR1 and B3, leading to catastrophic energy misallocation. These findings identify master transcriptional networks and stress-response pathways as pivotal indicators of embryogenic initiation efficiency, providing strategic targets for enhancing indica rice microspore culture technology."