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. 2025 Sep 29;15:33379. doi: 10.1038/s41598-025-18208-2

DNA TOP1α coordinates anther morphogenesis and fertility through gene regulatory networks in arabidopsis

Ibrahim Eid Elesawi 1,2,✉,#, Na Zhang 1,#, Ahmed M Hashem 1,3, Li Yao 1, Hesham M Abdullah 4, Mohamed Maher 2, Dalal Sulaiman Alshaya 5, Nora M Al Aboud 6, Salha Mesfer ALshamrani 7, Wael F Shehata 8, Abdallah A Hassanin 9,✉, Diaa Abd El-Moneim 10, Chunli Chen 1,✉
PMCID: PMC12480461  PMID: 41022959

Abstract

DNA topoisomerase 1 alpha (TOP1α) plays a critical role in plant development, yet its specific involvement in male reproduction is not well understood. Here, we show that TOP1α is essential for Arabidopsis anther development, and its absence leads to severe morphological and functional defects. The top1α1 mutant displays pleiotropic phenotypes, including early flowering and twisted petals, most notably the loss of two microsporangia. This loss stems from disrupted cell division in the L2 layer. Cytological analyses reveal aberrant pollen wall formation and reduced fertility in the mutant. RNA-seq data from top1α1 mutants identify over 2,000 differentially expressed genes, with significant downregulation of key anther development regulators such as SPL, AMS, and MS1, alongside genes involved in proteostasis and lipid metabolism. Furthermore, chromatin immunoprecipitation (ChIP) confirms TOP1α directly binds to the promoters of crucial genes like AG, MS1, and MYB80, suggesting a role in modulating transcriptional accessibility. All observed defects were rescued by complementation with gTOP1α-4HA, confirming the specificity of the top1α1 phenotype. Our findings establish TOP1α as a central coordinator of anther morphogenesis, functioning through intricate gene regulatory networks and chromatin remodeling. This work provides novel insights into the molecular mechanisms governing male reproductive development in plants.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-18208-2.

Keywords: Anther development, Arabidopsis, Gene expression, QPCR, DNA topoisomerase1, RNA-seq

Subject terms: Biotechnology, Molecular biology, Plant sciences

Introduction

Topoisomerases are essential enzymes that regulate DNA topology by altering DNA supercoiling and preventing topological stress during DNA replication, transcription, and recombination1,2. Beyond their fundamental role in DNA metabolism, topoisomerases are implicated in various cellular processes, including stress responses, such as the regulation of plant responses to light by TOP1α3. Based on the type of DNA break they induce, topoisomerases are classified into type I and type II enzymes. Type I topoisomerases, such as DNA topoisomerase 1 (TOP1), introduce transient single-stranded breaks in DNA. In contrast, type II topoisomerases, like DNA topoisomerase 2 (TOP2), generate transient double-stranded breaks. TOP2 plays a crucial role in transcriptional regulation by modulating nucleosome occupancy at gene promoters4. TOP1 is a key enzyme in DNA metabolism, exhibiting high expression during early embryonic development5. It also plays a vital role in regulating R-loop levels in plant chromatin, with TOP1 deficiency leading to R-loop accumulation6.

In Arabidopsis, the DNA topoisomerase 1α (TOP1α) gene was identified based on its sequence similarity to the fission yeast TOP1 gene7. The Arabidopsis genome encodes two type I topoisomerases: TOP1α and TOP1β, both located on chromosome 57. The essential roles of both enzymes in plant development are evident from the lethality of double homozygous mutants lacking both TOP1α and TOP1β7.

TOP1α plays crucial roles in various aspects of plant development, including organ initiation8,9, primary root development5, seed size10, and flowering time11. Disruptions in TOP1α function lead to aberrant plant architecture, characterized by serrated leaves, altered leaf angles, shortened internodes, and multiple flowers per node. Furthermore, TOP1α epigenetically regulates gene expression through H3K27me3 modifications and silences transposable elements12. Additionally, TOP1α is essential for proper meiotic progression, particularly homologous chromosome crossover formation13.

Anther development in flowering plants is a complex and precisely orchestrated process that culminates in the production of viable pollen, ensuring successful plant reproduction14. Anther development involves the coordinated differentiation of various cell types within a defined spatial and temporal framework15.

In Arabidopsis, anther development initiates from a single archesporial cell and progresses through 14 distinct stages. The anther primordium originates from three cell layers (L1-L3) within the floral meristem. Each layer contributes to specific anther structures: L1 forms the epidermis, L2 forms the parietal and sporogenous cells, and L3 forms the connective and vascular tissues. The parietal cells further differentiate into the tapetum, middle layer, and endothecium, which provide essential support and nutrients for developing pollen grains16,17.

While extensive research has been conducted on anther development, the genetic networks underlying early anther formation are still being elucidated. Key regulatory genes identified thus far include SPL/NZZ, EMS1, SERK1/2, and TPD118–20. The SPL transcription factor, downstream of the floral homeotic gene AGAMOUS, plays a crucial role in early anther development. Later stages of anther development are regulated by genes such as EMS1/EXS, SERK1, SERK2, and TPD1, which are involved in cell differentiation and fate determination within the anther21,22. Other genes implicated in early anther specification include LRR-RLKs such as BAM1-2, ER, ERL1-2, and MPK3-6, as well as transcription factors like DYT1, AMS, and MS123. These genes play essential roles in various aspects of anther development, including tapetum development, pollen cell wall formation, and the expression of HD-ZIP III genes in specific anther regions. This study investigates the role of TOP1α in anther development. We characterize the phenotypic alterations in anther architecture observed in top1α1 mutants, which carry a loss-of-function mutation in the TOP1α gene (AT5G55300). Our findings demonstrate that TOP1α plays a critical role in regulating the expression of genes essential for anther development and suggest that TOP1α is an integral component of the genetic network governing male reproductive organ formation in plants.

Results

Phenotypic analysis of top1α1 mutants

Phenotypic characterization of top1α1 mutants

Top1α1 mutants exhibited pleiotropic developmental defects compared to WT plants. Vegetative growth was characterized by serrated leaf margins, a phenotype also observed in top1α10 mutants. In addition, all flowers and inflorescences displayed a pronounced spiral morphology. Notably, top1α1 mutants exhibited accelerated growth and early flowering under both long-day and short-day conditions (Fig. 1).

Fig. 1.

Fig. 1

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The phenotype of top1α1 and transgenic plants relative to WT. (A) Representative images demonstrate an early flowering phenotype in top1α1 compared to WT and the complementary line (gTOP1α−4HA; top1α10), which rescued the null mutant phenotype under both short and long days. (B) Schematic representation of the TOP1α gene structure and T-DNA insertion loci in top1α10 and top1α1. Black boxes, gray boxes and black lines represent exons, untranslated regions, and introns respectively. top1α10 contained a T-DNA insertion in the first intron and served as the background for the complementary line. top1α1 included a T-DNA insertion in the eighth intron. (C) WT and (D) top1α1. Scale bar =1mm.

Floral defects in top1α1 mutants

Flowers in top1α1 mutants were smaller than WT flowers, with twisted petals (Fig. 2A) and aberrantly shaped anthers (Fig. 2B). Microscopic analysis revealed severe defects in anther development. While WT anthers typically develop four functional microsporangia filled with viable pollen grains (Fig. 2C, D), top1α1 anthers were smaller and displayed a significant reduction in microsporangia number and function. The inner microsporangia were often shrunken and devoid of pollen grains (Fig. 2C, D, Figure S1). Furthermore, the few pollen grains produced in top1α1 mutants often exhibited morphological abnormalities (Fig. 3A, B, C).

Fig. 2.

Fig. 2

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Anther phenotype of top1α1 anthers relative to WT. (A) The figure shows the open flowers in WT and top1α1, highlighting the difference between the WT and top1α flowers (B) Compares the reproductive organs of WT and top1α1, illustrating the extent of anthers deformation in the absence of TOP1α function. (C) Anther phenotype under SEM in both WT and top1α1. (D) Pollen viability was observed with Alexander stain (Scale bar = 100 μm).

Fig. 3.

Fig. 3

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Scanning electron microscope analysis of WT and top1α1 mutant anthers. (A, B, C) WT anther containing viable pollen grains. (D) Pollen from the WT anther. (E, F) Exine layer of the WT pollen. (a, b, c) top1α1 anther containing failed pollen grains. (d) Pollen from the top1α1 mutant anther, (e, f) Exine layer of the top1α1 mutant pollen. The sample size (n ≥ 5 anthers per genotype).

Pollen grain abnormalities and reduced fertility

Scanning electron microscopy (SEM) of top1α1 (Fig. 3a-f) pollen grains revealed significant defects in the exine. These abnormalities were in stark contrast to the uniform, well-defined exine of wild-type (WT) pollen. Compared to the consistent and evenly-spaced ridges of WT pollen (Fig. 3A-F), top1α1 pollen displayed a chaotic and non-uniform ridge pattern, with irregular thickening. This aberrant deposition leads to bulging, distorted ridges that compromise the pollen grain’s structural integrity.

The spaces between the ridges, known as lacunae, were also significantly compressed. In healthy pollen, these spaces contribute to the exine’s robustness. Their compression in top1α1 mutants indicates a failure in the proper expansion of the exine framework during development.

The most dramatic defect was the presence of“missing areas”or gaps in the exine. In these regions, the sporopollenin framework was completely absent, leaving the inner pollen wall, the intine, exposed. Such gaps represent a complete failure in exine synthesis, severely compromising the pollen grain’s ability to withstand environmental stress and leading to a loss of viability.

These morphological defects have direct functional consequences. The compromised exine structure in top1α1 mutants leads to a significant reduction in pollen viability (Figure S2). This reduced pollen viability directly impacts the fertility of top1α1 plants, resulting in fewer successful fertilization events. This leads to shorter siliques and a significant reduction in the number of seeds per silique, establishing a clear link between the microscopic defects in pollen wall formation and the macroscopic phenotype of reduced seed yield (Fig. 4).

Fig. 4.

Fig. 4

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Silique length and number of seeds per silique. (A) Silique lengths of WT and top1α1 are compared. (B) Quantitative analysis comparing silique length in WT and top1α1. (C) Seeds within WT and top1α1 siliques. (D) Quantitative analysis comparing seed number in WT and top1α1. *** denotes P<0.001.

Complementation analysis confirms the role of TOP1α in anther development

Complementation assays using a gTOP1α−4HA construct in top1α10 mutants confirmed the role of TOP1α in anther development. The complemented lines exhibited normal microsporangia and anther morphology (Figures S3A and B), demonstrating that the observed phenotypes in top1α1 mutants are directly attributable to the loss of TOP1α function.

The analysis of the complemented lines (gTOP1α−4HA in top1α10 background) was performed on n = 12 independent transgenic lines. All 12 lines showed a full rescue of the anther phenotype. For the representative images shown in Figure S3, anthers from at least 10 individual plants per line were examined, with consistent results.

top1α1 anther defects in microsporangia formation

To investigate the precise developmental stages at which anther abnormalities occur in the top1α1 mutant, we meticulously examined anther development in both top1α1 and wild-type (WT) plants. Cross-sectional analyses of anther primordia at various developmental stages (Fig. 5) were conducted. During the initial stages of anther development (stages 1 and 2), no discernible differences were observed between top1α1 and WT anthers, with all three cell layers (L1, L2, and L3) correctly established (Fig. 5A, B, a, b). However, distinct developmental aberrations emerged in top1α1 anthers from stage 3 onwards. In WT, periclinal and anticlinal divisions within the L2 layer at the four corners of the anther primordium at stage 3 gave rise to four distinct microsporangial primordia (Fig. 5C). This pattern continued throughout subsequent stages, culminating in the formation of four functional microsporangia and the production of viable pollen grains (Fig. 5D-H). In contrast, top1α1 anthers exhibited a significant reduction in microsporangia formation. At stage 5, while WT anthers displayed four well-defined microsporangia, top1α1 anthers only developed two (Fig. 5E, e). This reduction in microsporangia persisted throughout later stages of development, with top1α1 anthers ultimately producing microspores only within two outer lobes, whereas WT anthers formed microspores in all four lobes (Fig. 5H, h).

Fig. 5.

Fig. 5

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Loss of microsporangia in top1α1 stamen primordia. Paraffin-embedded cross-sections of anthers at different stages, stained with toluidine Blue. (A-H) Different stages in WT anthers, and (a-h) corresponding stages in top1α1 anthers". L1, L2, and L3, the three cell-layers in stamen primordia; Ar, archesporial cell; E, epidermis; En, endothecium; M, middle layer; SC, sporogenous cells; T, tapetum; Tds, tetrads; PMC, pollen mother cell; Msp, microspore; PG, pollen grain. n ≥ 15 anther primordia per stage per genotype. Bars =50 μm.

Ubiquitous expression of TOP1α in arabidopsis

To investigate the temporal and spatial expression patterns of TOP1α during anther development, we generated a gTOP1α-GUS fusion construct. This construct comprised a 5.6-kb TOP1α genomic region fused to the GUS reporter gene, cloned into the pHY107 vector. Histochemical analysis of gTOP1α-GUS activity revealed ubiquitous expression of TOP1α in various Arabidopsis tissues throughout the vegetative stage and floral transition, with notably strong expression in the meristem zone (Fig. 6).

Fig. 6.

Fig. 6

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Transgenic gTOP1α-GUS Plants Stained for GUS Activity. (A, B) These images show the expression of the TOP1A during the seedling stage in the shoot apical meristem. (C, D) GUS staining of a representative gTOP1α-GUS line at the flowering time.

Expression of anther development-related genes in top1α1

To investigate the impact of TOP1α on anther development, we performed gene expression analysis in top1α1 mutant and wild-type (WT) plants. RNA was extracted from pre-blooming inflorescences, and cDNA was synthesized for quantitative real-time PCR (qRT-PCR) analysis (Fig. 7). qRT-PCR revealed significant downregulation of several genes crucial for anther development in top1α1 compared to WT. These included genes involved in cell differentiation (e.g., AG, SPL, EMS1, SRPK1), LRR-RLK signaling (e.g., BAM1, SERK1, ER, LER1, LER2), and other key regulatory pathways (e.g., AMS1, DYT1, MS1, MS2, ROXY1, ROXY2, MYB33, HYL1). While most members of the HD-ZIP III family showed comparable expression levels, slight downregulation of REV and CAN was observed in top1α.

Fig. 7.

Fig. 7

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DqRT-PCR analysis of candidate genes involved in anther development. The expression levels of candidate genes in WT and top1α1 mutants are shown. Error bars represent the standard deviation of three independent biological replicates. Actin 8 was used as a housekeeping gene for normalization. *** denotes P < 0.001, ** denotes P < 0.05, and * denotes P < 0.05.

RNA-seq analysis of differentially expressed genes

To understand the molecular impact of TOP1α deficiency on gene expression, we performed RNA-Seq analysis of top1α1 mutants (Fig. 6). Compared WT plants, 2,056 genes exhibited significantly altered expression levels in top1α1 mutants. Of these, 1,617 genes were downregulated, while 439 genes were upregulated (Fig. 8 and Table S2).

Fig. 8.

Fig. 8

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Differential Gene Expression Analysis in top1α1 Mutants reveals profound alterations in anther and pollen development. (A) MA plots represent log2 fold change (FC) on the y-axis and average expression on the x-axis, highlighting significant gene expression changes in the top1α1 mutant compared to WT. (B) Venn diagrams illustrate the number of Differentially Expressed Genes (DEGs) in the top1α1 mutant relative to WT, categorized as upregulated, downregulated, or showing no significant changes. (C) Heatmaps display expression changes (log2 FC) between the topα1 mutant and WT, with clustering of three biological replicates in each group and clustering of DEGs.

MA plots (Fig. 8A) visually represent the log2 fold change (FC) in gene expression against the average expression level, effectively highlighting significant gene expression changes in top1α1 mutants. A Venn diagram (Fig. 8B) illustrates the distribution of differentially expressed genes (DEGs) in top1α1 relative to WT, categorizing them as upregulated, downregulated, or unchanged. Heatmaps (Fig. 8C) provide a visual representation of the log2 FC values for DEGs between top1α1 and WT, including clustering of biological replicates, further emphasizing the substantial impact of TOP1α on gene expression regulation. To gain insights into the biological functions of the DEGs, we performed Gene Ontology (GO) enrichment analysis (Fig. 9 and Tables S3 and S4). GO terms were categorized into three major ontologies: biological processes (Fig. 9A), molecular functions (Fig. 9B), and cellular components (Fig. 9C). Statistical significance was determined using a p-value cutoff of 0.05, and the DAVID bioinformatics resource was employed for GO enrichment analysis.

Fig. 9.

Fig. 9

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Gene Ontology (GO) enrichment analysis of DEGs down-regulated in the top1α1 mutant relative to WT24,25. (A) GO enrichment analysis of DEGs related to biological processes. (B) GO enrichment analysis is focused on DEGs associated with molecular functions. (C) GO enrichment analysis concerning cellular components, localization, or compartments.

Direct binding of TOP1α to anther development-related genes

To investigate whether TOP1α directly interacts with genes involved in anther development, we performed chromatin immunoprecipitation (ChIP) followed by quantitative PCR (qPCR) experiments. We examined the binding of TOP1α to the loci of key anther development genes, including AG, AMS, CAL5, MPK3, MS1, MS2, ROXY2, SPL, MYB99, MYB80, NST1, SHN3, ERF6, ACS6, and WUS1, in the top1α10 top1α10::gTOP1α−4HA transgenic line, which rescues the morphological defects observed in top1α1 mutants (Fig. 10A-C). In young flower buds, ChIP-qPCR analysis demonstrated enrichment of TOP1α at the loci of all examined genes in the anti-HA immunoprecipitated samples from the top1α10 top1α10::gTOP1α−4HA transgenic line compared to non-antibody controls. Interestingly, TOP1α binding was significantly higher in the coding regions of AG, MPK3, MS1, SPL, ERF6, and ACS6 compared to their respective promoter regions.

Fig. 10.

Fig. 10

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ChIP-qPCR analysis reveals TOP1α binding at the AG, MS1, and MYB80 genomic regions, potentially enhancing transcription. (A) A schematic diagram illustrates the designed ChIP-qPCR primers regions. P1 and P2 are located in the promoter region, with P1 further away from the Transcription Start Site (TSS) than P2. Primer P is located downstream of the coding region. (B and C) ChIP-qPCR analysis of TOP1α−4HA binding to genomic fragments in top1α10::gTOP1α−4HA young flower buds. Error bars represent standard error, with different letters denoting significant differences (LSD test, p ≤ 0.01).

Discussion

DNA TOP1α is a crucial enzyme involved in plant development, primarily functioning as a transcriptional regulator8,13. In Arabidopsis thaliana, loss-of-function of TOP1α in the top1α1 mutant leads to early flowering compared to WT and complemented lines. TOP1α regulates flowering time by controlling the expression of FLOWERING LOCUS C (FLC) and its homologs, key repressors of flowering, through mechanisms involving transcriptional machinery and histone modifications8,26.

This study investigated the role of TOP1α in anther development. While top1α1 stamens appear indistinguishable from WT stamens during early stages (Stage 2), morphological and size differences become evident in Stage 3, suggesting a crucial role for TOP1α in later stages of anther development. Similar to previous observations27, the TOP1α mutation has a greater impact on the development of outer microsporangia compared to inner microsporangia.

Gene expression analysis revealed significant downregulation of several genes essential for anther development in top1α1 mutants, including those involved in anther cell differentiation: SPL, EMS, SERK228–30 stamen cell differentiation and tapetum development: ER, ERL1-2, BAM1, BAM231, anther development: AMS, DYT1, ROXY2, MS1, MS2, stomium, vascular bundle, and middle region development: REV, CAN, HP8 (HD-ZIP III family). These findings suggest that TOP1α may act upstream of DYT1, AMS, TDF1, and MS188, aligning with previous transcriptome analyses of dyt1, ams, and ms188 mutants, which showed no effect on TOP1α expression32.

Previous research has demonstrated that TOP1α regulates AG by controlling H3K27me3 levels during the floral transition26,33. investigated gene expression changes in top1α mutant inflorescences and explored the role of nucleosome occupancy and PcG complex targets. We propose that TOP1α influences floral and anther differentiation and development by modulating nucleosome density and H3K27me3 levels on AG and other genes, including SLP, DYT1, MS1, MS2, AMS, EMS1, and others. Further studies are required to elucidate the direct or indirect nature of TOP1α regulation on these genes.

ChIP-qPCR assays revealed that TOP1α binds to the promoter regions of many genes more frequently than the coding regions, suggesting that TOP1α primarily regulates gene expression by binding to promoter regions.

This study provides comprehensive insights into the multifaceted roles of TOP1α in plant development, particularly in reproductive organ formation. The observed downregulation of genes involved in a wide range of biological processes, including flavonoid and phenylpropanoid biosynthesis, auxin and jasmonic acid biosynthesis, DNA binding, kinase activity, and transporter activity, underscores the pleiotropic nature of TOP1α function5,26,30.

The significant downregulation of genes associated with pollen development, cell differentiation, and cell wall organization directly correlates with the observed defects in pollen development and anther cell specification in the top1α1 mutant. These findings suggest that TOP1α modulate gene expression via the modulating chromatin accessibility. The study conducted by Takahashi, et al.34 revealed that TOP1α is crucial for maintaining the regular pattern of organ initiation in plants. Furthermore, the lethality of a top1α mutant with reduced TOP1β expression indicates that topo I activity is essential for early plant development.

A similar study by Liu, et al.35 elucidated a specific role for the DNA topoisomerase TOP1α in plant development by demonstrating its involvement in Polycomb Group (PcG) protein function. Specifically, TOP1α is required for the deposition of the repressive histone mark H3K27me3 at the stem cell maintenance gene WUSCHEL (WUS), leading to its transcriptional repression in Arabidopsis floral stem cells. This requirement extends to other PcG target genes. Intriguingly, TOP1α is implicated in both the repression of certain PcG target genes and the expression of others. The proposed mechanism underlying these seemingly opposing effects posits that TOP1α reduces nucleosome density, potentially facilitating the binding of factors that either recruit PcG, as observed with AGAMOUS at WUS, or counteract PcG-mediated repression.

The downregulation of genes involved in proteasome regulation indicates that TOP1α may influence protein degradation pathways, with potential consequences for cellular homeostasis and the removal of misfolded or damaged proteins.

The upregulation of certain genes in the top1α1 mutant may represent compensatory mechanisms to mitigate the effects of TOP1α loss. The upregulation of genes involved in hypoxia response, defense against pathogens, and stress responses indicates that TOP1α may also play a role in these processes. The upregulation of genes involved in the cellular response to low oxygen levels suggests that the loss of TOP1α function may trigger hypoxic conditions. Additionally, the upregulation of defense response genes implies that TOP1α may play a role in modulating pathogen defense pathways.

Materials and methods

Plant materials and growth conditions

All plants used in this study were A. thaliana (Col-0). The top1α1 containing a T-DNA insertion in the eighth intron was generated by36. Similarly, the top1a10 (SALK_013164) harbors a T-DNA insertion in the first intron.

To generate transgenic lines gTOP1a-4HA and gTOP1α-GUS in the top1α10 mutant background, we employed the Agrobacterium tumefaciens-mediated floral dip method33. The gTOP1a-4HA line incorporates a 5.6-kb genomic fragment of TOP1α, encompassing 1.3 kb of the upstream 5’ regulatory region, the complete coding sequence, and a C-terminal 4xHA epitope tag fused in-frame within an intron. To generate the gTOP1α-GUS line, we cloned the identical 5.6-kb genomic fragment into the pHY107 vector. Transgenic lines were selected on soil using Basta herbicide containing phosphinothricin. Plants were maintained in a growth chamber under long-day conditions (16 hours light/8 hours dark) at approximately 22°C.

Pollen grains and mature anther staining

To assess pollen viability and mature anther phenotypes, we stained pollen grains with Alexander’s stain following the manufacturer’s protocol37. This enabled differentiation between viable (magenta-red) and aborted (blue or green) pollen grains40. Briefly, flower buds at stage 12 of anther development were collected and fixed in Carnoy’s solution (ethanol:chloroform:acetic acid, 6:3:1 v/v) for at least two hours. After fixation and dissection under a binocular microscope, individual anthers were incubated with Alexander’s stain for 30 minutes at 25°C. Subsequently, the anthers were mounted on slides and visualized using an Olympus BX53F microscope equipped with a Canon digital camera38.

Paraffin histology and histochemistry

The flower buds were collected and directly fixed by Formaldehyde-Acetic Acid (FAA) solution (3.7%, formaldehyde, 50%, acetic acid, and 5% ethanol) under a vacuum for about 15 min, as described by38,39. Briefly, the fixative buffer was removed and the tissues were incubated with fresh fixative buffer overnight. The tissues were then dehydrated through a graded ethanol series (70%, 85%, 95%) containing 1% eosin Y for 1 hour each, followed by two incubations in pure alcohol for 1 hour each. Added tiny paraffin to a glass bottle that contains chloroform. incubated bottles at 37℃ for 2-3 days, added a little paraffin every six hours if the paraffin dissolved completely, and made sure samples were infiltrated by tiny paraffin. The samples were then transferred from the glass bottle to small embedding molds and embedded in paraffin wax using a paper boat. Immersed samples in a serial concentration of wax with chloroform: 50 % (2h), 75% (2h), pure wax (1h), pure wax (1h), and pure wax (1h); then the samples were embedded in paraffin with a paper box. The cross-sections were prepared with a rotary microtome, thickness of sections of 0.5 μm. The sections were stained by Toluidine Blue O buffer for one minute, observed, and then photographed under Olympus BX53F microscope (Olympus, Tokyo, Japan) with a Canon digital camera (Canon, Tokyo, Japan).

Scanning electron microscopy (SEM)

Anther development in wild-type and mutant plants was examined using scanning electron microscopy (SEM). We analyzed pollen grains from a minimum of 5 different anthers per genotype (n ≥ 5 anthers per genotype). Flower buds were collected and fixed overnight at 4 °C in FAA fixative. Following fixation, samples were dehydrated through a graded ethanol series and subsequently critical point dried using liquid CO2. Individual anthers from flowers at comparable developmental stages were carefully dissected and mounted for observation under a Hitachi SU8010 ultra-high-resolution SEM at an accelerating voltage of 10 kV, the magnification power varied from 160x to 15000x38.

Histochemical localization of GUS activity

To investigate the temporal and spatial expression patterns of TOP1α during anther development, a GUS staining assay was performed40,41. Inflorescences were incubated overnight at 37 °C in GUS staining buffer (50 mM sodium phosphate buffer, pH 7.0, containing 0.2% Triton X-100, 10 mM potassium ferrocyanide, 10 mM potassium ferricyanide, and 1 mM X-gluc). Following incubation, plant materials were cleared using an alcohol dilution series. GUS activity was visualized using an Olympus Macro zoom stereo microscope equipped with a Canon digital camera.

Quantitative real-time PCR analyses

Total RNA was extracted from inflorescences prior to stage 12 using TRIzol reagent (Invitrogen). Subsequently, 3 μg of the extracted RNA was employed to synthesize the first-strand cDNA using 200 units of M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was then performed using an ABI StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Green Master Mix (Roche). Each sample was evaluated in at least three replicates. Gene-specific primers for the target genes are listed in Table S1.

RNA-seq analysis

Total RNA was extracted from young flower buds of six-week-old wild-type (WT) and top1a1 plants using the RNAprep Pure Plus Kit. Genomic DNA was removed with DNase I. RNA-seq was performed on three biological replicates. Raw reads were aligned to the TAIR10 Arabidopsis genome using HISAT242. Gene expression levels were quantified as transcripts per million mapped reads (TPM). Differentially expressed genes (DEGs) were identified using DESeq243, with a significance threshold of FDR-adjusted P-value ≤ 0.05 and a fold change (log2FC) > 1.5 or < −1.5 (Table S2). The three biological replicates were used to calculate the false discovery rate.

Functional annotation and enrichment analysis

To functionally characterize the differentially expressed genes (DEGs) and gain biological insights into their roles, we employed enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov/tools.jsp). This analysis identified enriched Gene Ontology (GO) terms associated with the DEGs. To ensure the accuracy and relevance of these terms, we further verified them using clusterProfiler, a bioinformatics toolkit specifically designed for analyzing high-throughput gene expression data44. Lists containing Arabidopsis thaliana gene identifiers for up and downregulated DEGs (Table S2) were uploaded to DAVID and analyzed using the functional annotation tool. GO terms related to biological process, molecular function, and cellular components, as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were queried for statistically significant enrichment within the DEG lists compared to the whole Arabidopsis genome background. This enrichment analysis identifies GO terms and KEGG pathways that are statistically overrepresented in the DEG datasets compared to the whole Arabidopsis genome background.

For the analysis, the functional annotation clustering option was applied with the following parameters: Gene Ontology and Pathways databases selected, medium classification stringency, enrichment thresholds of EASE score/P-value < 0.,1, and minimum enrichment score > 1.5. Benjamini-Hochberg multiple testing correction was used to control the false discovery rate. Significantly enriched terms meeting the threshold criteria were recorded along with enrichment scores, P-values Benjamini-adjusteddd P-values, and gene counts (Tables S3 and S4). This allowed functional categorization of the DEGs and identification of biological processes, molecular functions, cellular compartments, and pathways most perturbed as a consequence of TOP1α loss in the mutant.

ChIP-qPCR analysis

ChIP (chromatin immunoprecipitation) assays were performed following an integrated method45 and utilized ChIP-Grade Protein G beads (Cell Signaling Technology, Cat#: 9006). Six-week-old top1a10::gTOP1a-4HA young flower buds were fixed with 1% (wt/vol) formaldehyde and subsequently stopped with 2M glycine. Nuclei were lysed to obtain 200–800 bp chromatin fragments. The HA antibody (Cell Signaling Technology, Cat#: 3724) was used for ChIP. 5M sodium chloride (NaCl) was then added to the input and the product from immunoprecipitation and incubated overnight at 65ºC to reverse the cross-linking. The chromatin fragments were then treated with 0.5 M EDTA pH 8.0, 1 M Tris-HCl pH 6.5, and proteinase K. Following phenol-chloroform-isoamyl alcohol (25:24:1) extraction, the purified DNA, along with input and antibody-bound DNA samples, underwent qPCR analysis in triplicate. qPCR was performed using the TransStart® Tip Green qPCR SuperMix (TRANSGEN, Cat#: AQ141-04) in the lightcycle 480Ⅱ real-time system (Roche). To ensure reproducibility, three biological replicates were conducted. All the primers used for ChIP-qPCR are listed in Table S1.

Statistical analysis

In all the analyses, unless mentioned otherwise, the number of replicates (n) and the standard error (SE) are shown for most measurements. The data were statistically analyzed using ANOVA (P < 0.05). ANOVA was performed considering the degrees of freedom (df) for each analysis.

Conclusions

In conclusion, our study unveils a novel and critical role for DNA topoisomerase 1 alpha (TOP1α) in Arabidopsis anther development, demonstrating that its absence in the top1α1 mutant leads to the loss of two microsporangia, likely due to the reduced expression of key genes involved in cell differentiation and anther formation. Comprehensive RNA-seq analysis revealed widespread transcriptional changes, affecting pathways crucial for pollen development, proteasome regulation, reproductive processes, growth, and lipid metabolism. These findings provide significant new insights into the complex gene regulatory network controlled by TOP1α during male reproductive organogenesis in plants, highlighting its unexpected importance for proper anther architecture and paving the way for future investigations into the precise molecular mechanisms involved.

Supplementary Information

Supplementary Information 1. (811.5KB, docx)
Supplementary Information 2. (60.2KB, docx)
Supplementary Information 3. (1.8MB, docx)
Supplementary Information 4. (290.8KB, xlsx)
Supplementary Information 5. (224.8KB, xlsx)
Supplementary Information 6. (33.9KB, xlsx)
Supplementary Information 7. (31.4KB, xlsx)

Acknowledgments

The authors extend their appreciation to the National Natural Science Foundation of China (No.31970525), the Advanced Foreign Experts Project (G2023157014L), and the Cultivating Fund Project of Hubei Hongshan Laboratory (2022hspy002). Also the authors would like to acknowledge the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R465), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. and the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (Project No. KFU253078), for supporting this work.

Author contributions

Conceptualization, I.E.E., L.Y., N.Z., and C.C.; methodology, I.E.E., L.Y., N.Z., and C.C.; software, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C.; validation, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C.; formal analysis, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C.; investigation, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C.; data curation;, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C. writing—original draft preparation, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C.; writ-ing—review and editing; visualization, I.E.E., N.Z., A.M.H., L.Y., H.M.A., M.M., D.S.A., N.M.A., S.M.A., W.F.S., A.A.H., D.A-E., and C.C.; supervision, I.E.E. and C.C.; project administration, I.E.E. and C.C.; funding acquisition, D.S.A., I.E.E., and C.C. All authors have read and agreed to the pub-lished version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No.31970525), the Advanced Foreign Experts Project (G2023157014L), and the Cultivating Fund Project of Hubei Hongshan Laboratory (2022hspy002). This work was also supported by the Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R465), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, and the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Project No. KFU253078].

Data availability

The RNA-seq data will be made available to readers upon request from the corresponding authors. The accession number is currently under process.

Declarations

Conflicts of Interest

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ibrahim Eid Elesawi and Na Zhang are authors contributed equally.

Contributor Information

Ibrahim Eid Elesawi, Email: ibrahimeid@zu.edu.eg.

Abdallah A. Hassanin, Email: asafan@zu.edu.eg

Chunli Chen, Email: chenchunli@mail.hzau.edu.cn.

References

  • 1.Nitiss, K. C., Bandak, A., Berger, J. M. & Nitiss, J. L. Genome Instability Induced by Topoisomerase Misfunction. Int. J. Mol. Sci.25, 10247. 10.3390/ijms251910247 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Dasgupta, T., Ferdous, S. & Tse-Dinh, Y.-C. Mechanism of Type IA Topoisomerases. Molecules25, 4769 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ma, Y. et al. Recent advances in response to environmental signals during Arabidopsis root development. Plant Physiol Bichem.215, 109037. 10.1016/j.plaphy.2024.109037 (2024). [DOI] [PubMed] [Google Scholar]
  • 4.Vega, M. et al. Topoisomerase 1 facilitates nucleosome reassembly at stress genes during recovery. Nucleic Acids Res.51, 12161–12173. 10.1093/nar/gkad1066 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Zhang, H. et al. TOP1α suppresses lateral root gravitropism in Arabidopsis. Plant Signal. Behav.17, 2098646. 10.1080/15592324.2022.2098646 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Patel, P. S., Krishnan, R. & Hakem, R. Emerging roles of DNA topoisomerases in the regulation of R-loops. Mutat. Res. Genet. Toxicol. Environ. Mutagen.876–877, 503450. 10.1016/j.mrgentox.2022.503450 (2022). [DOI] [PubMed] [Google Scholar]
  • 7.Gong, X., Shen, L., Peng, Y. Z., Gan, Y. & Yu, H. DNA Topoisomerase Iα Affects the Floral Transition. Plant Physiol.173, 642–654. 10.1104/pp.16.01603 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Peng, D. et al. Functional characterisation of BnaA02.TOP1α and BnaC02.TOP1α involved in true leaf biomass accumulation in Brassica napus L. Plant J.10.1111/tpj.17054 (2024). [DOI] [PubMed] [Google Scholar]
  • 9.Gu, N., Chen, C., Kabeya, Y., Hasebe, M. & Tamada, Y. Topoisomerase 1α is required for synchronous spermatogenesis in Physcomitrium patens. New Phytol.234, 137–148. 10.1111/nph.17983 (2022). [DOI] [PubMed] [Google Scholar]
  • 10.Li, C. et al. TOP1α, UPF1, and TTG2 regulate seed size in a parental dosage–dependent manner. PLOS Biol.18, e3000930. 10.1371/journal.pbio.3000930 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zong, W. et al. DEK domain-containing proteins control flowering time in Arabidopsis. New phytol.231, 182–192. 10.1111/nph.17366 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Inagaki, S., Takahashi, M., Takashima, K., Oya, S. & Kakutani, T. Chromatin-based mechanisms to coordinate convergent overlapping transcription. Nature Plants7, 295–302. 10.1038/s41477-021-00868-3 (2021). [DOI] [PubMed] [Google Scholar]
  • 13.Elesawi, I. E. et al. The role of DNA topoisomerase 1α (AtTOP1α) in regulating arabidopsis meiotic recombination and chromosome segregation. PeerJ12, e17864. 10.7717/peerj.17864 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wilson, Z. A. & Zhang, D.-B. From Arabidopsis to rice: pathways in pollen development. J. Exp. Bot.60, 1479–1492. 10.1093/jxb/erp095 (2009). [DOI] [PubMed] [Google Scholar]
  • 15.Goldberg, R. B., Beals, T. P. & Sanders, P. M. Anther development: basic principles and practical applications. Plant Cell5, 1217–1229. 10.1105/tpc.5.10.1217 (1993). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Åstrand, J., Knight, C., Robson, J., Talle, B. & Wilson, Z. A. Evolution and diversity of the angiosperm anther: trends in function and development. Plant Reprod.34, 307–319. 10.1007/s00497-021-00416-1 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Marchant, D. B. & Walbot, V. Anther development—The long road to making pollen. Plant Cell34, 4677–4695. 10.1093/plcell/koac287 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Xue, J.-S. et al. Development of the Middle Layer in the Anther of Arabidopsis. Front. Plant Sci.10.3389/fpls.2021.634114 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zheng, Y. et al. Auxin guides germ-cell specification in Arabidopsis anthers. Proc. Natl. Acad. Sci.118, e2101492118. 10.1073/pnas.2101492118 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wang, D. et al. BRI1 EMS SUPPRESSOR1 genes regulate abiotic stress and anther development in wheat (Triticum aestivum L.). Front. Plant Sci.10.3389/fpls.2023.1219856 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Yu, Y. et al. PXL1 and SERKs act as receptor–coreceptor complexes for the CLE19 peptide to regulate pollen development. Nat. Commun.14, 3307. 10.1038/s41467-023-39074-4 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Soltabayeva, A. et al. Receptor-like Kinases (LRR-RLKs) in Response of Plants to Biotic and Abiotic Stresses. Plants11, 2660. 10.3390/plants11192660 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Cai, Y. et al. The HD-ZIP IV transcription factor GL2-LIKE regulates male flowering time and fertility in cucumber. J. Exp. Bot.71, 5425–5437. 10.1093/jxb/eraa251 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kanehisa, M., Sato, Y., Kawashima, M., Furumichi, M. & Tanabe, M. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res.44, D457–D462. 10.1093/nar/gkv1070 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res.28, 27–30. 10.1093/nar/28.1.27 (2000). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Zhong, P., Li, J., Luo, L., Zhao, Z. & Tian, Z. TOP1α regulates FLOWERING LOCUS C expression by coupling histone modification and transcription machinery. Development10.1242/dev.167841 (2019). [DOI] [PubMed] [Google Scholar]
  • 27.Sanders, P. M. et al. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant Reprod.11, 297–322 (1999). [Google Scholar]
  • 28.Hord, C. L. H., Chen, C., DeYoung, B. J., Clark, S. E. & Ma, H. The BAM1/BAM2 Receptor-Like Kinases Are Important Regulators of Arabidopsis Early Anther Development. Plant Cell18, 1667–1680. 10.1105/tpc.105.036871 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Liu, J., Li, W., Wu, G. & Ali, K. An update on evolutionary, structural, and functional studies of receptor-like kinases in plants. Front. Plant Sci.10.3389/fpls.2024.1305599 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Biswas, R. & Chaudhuri, S. The tale of tapetum: from anther walls to pollen wall. Nucleus10.1007/s13237-024-00510-5 (2024). [Google Scholar]
  • 31.Alam, I., Batool, K., Huang, Y., Liu, J. & Ge, L. Developing Genetic Engineering Techniques for Control of Seed Size and Yield. Int. J. Mol. Sci.23, 13256. 10.3390/ijms232113256 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wei, S. & Ma, L. Comprehensive Insight into Tapetum-Mediated Pollen Development in Arabidopsis thaliana. Cells12, 247. 10.3390/cells12020247 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Gong, X., Shen, L., Peng, Y. Z., Gan, Y. & Yu, H. DNA topoisomerase Iα affects the floral transition. Plant Physiol.173, 642–654. 10.1104/pp.16.01603 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Takahashi, T., Matsuhara, S., Abe, M. & Komeda, Y. Disruption of a DNA Topoisomerase I Gene Affects Morphogenesis in Arabidopsis. Plant Cell14, 2085–2093. 10.1105/tpc.001925 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Liu, X. et al. DNA Topoisomerase I Affects Polycomb Group Protein-Mediated Epigenetic Regulation and Plant Development by Altering Nucleosome Distribution in Arabidopsis. Plant Cell26, 2803–2817. 10.1105/tpc.114.124941 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Matsuhara, S., Jingu, F., Takahashi, T. & Komeda, Y. Heat-shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T-DNA insertions. Plant J.22, 79–86. 10.1046/j.1365-313x.2000.00716.x (2000). [DOI] [PubMed] [Google Scholar]
  • 37.Alexander, M. P. Differential Staining of Aborted and Nonaborted Pollen. Stain. Technol.44, 117–122. 10.3109/10520296909063335 (1969). [DOI] [PubMed] [Google Scholar]
  • 38.Chang, F., Zhang, Z., Jin, Y. & Ma, H. in Flower Development 203-216 (Springer, 2014).
  • 39.Jacobowitz, J. R., Doyle, W. C. & Weng, J.-K. PRX9 and PRX40 are extensin peroxidases essential for maintaining tapetum and microspore cell wall integrity during Arabidopsis anther development. Plant Cell31, 848–861 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant mol. Boil. Rep.5, 387–405 (1987). [Google Scholar]
  • 41.Sessions, A., Weigel, D. & Yanofsky, M. F. The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J.20, 259–263. 10.1046/j.1365-313x.1999.00594.x (1999). [DOI] [PubMed] [Google Scholar]
  • 42.Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods12, 357–360. 10.1038/nmeth.3317 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol.11, R106. 10.1186/gb-2010-11-10-r106 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Yu, G., Wang, L. G., Han, Y. & He, Q. Y. clusterProfiler: an R package for comparing biological themes among gene clusters. Omics:J. Integr. Biol.16, 284–287. 10.1089/omi.2011.0118 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Zhang, P. et al. Rice and Arabidopsis homologs of yeast CHROMOSOME TRANSMISSION FIDELITY PROTEIN 4 commonly interact with Polycomb complexes but exert divergent regulatory functions. Plant Cell33, 1417–1429 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information 1. (811.5KB, docx)
Supplementary Information 2. (60.2KB, docx)
Supplementary Information 3. (1.8MB, docx)
Supplementary Information 4. (290.8KB, xlsx)
Supplementary Information 5. (224.8KB, xlsx)
Supplementary Information 6. (33.9KB, xlsx)
Supplementary Information 7. (31.4KB, xlsx)

Data Availability Statement

The RNA-seq data will be made available to readers upon request from the corresponding authors. The accession number is currently under process.

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