Preamble

Production of unreduced microspores in Arabidopsis flowers cultivated in culture medium suggests a role of sucrose in facilitating meiotic cytokinesis

Huiqi Fu¹,#, Yuting Chen¹,#, Xueying Cui¹, Huishan He¹, Jingru Wang², Chong Wang³, Ziming Ren²,, Bing Liu¹,,‡

¹Arameiosis Lab, South-Central Minzu University, Wuhan 430074, China.
²Department of Landscape Architecture, Zhejiang Sci-Tech University, Hangzhou 310018, China.
³Shanghai Key Laboratory of Plant Molecular Sciences, Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China.

These authors contributed equally to this study and thus share the first authorship.

*Correspondence: B.L. (arameiosis@163.com); Z.R (zimingren@zju.edu.cn).
‡Senior author.

Short title: Sucrose facilitates male meiotic cytokinesis.

One-sentence summary: Arabidopsis flowers cultivated in culture medium produce unreduced microspores due to interfered meiotic cytokinesis, which is partially rescued by increased sucrose supply.

Abstract

Live-imaging microscopy technology has been increasingly applied for meiosis study in plants, which largely relies on the establishment of a healthy ex vivo culture system for inflorescences to ensure that the captured chromosome dynamics approach the natural features of meiosis. Here, we report that Arabidopsis thaliana flowers cultivated in a culture medium (CCM) composed of half-strength Murashige and Skoog basal salt, MES, myo-inositol, sucrose, and agar produce diploid microspores due to the occurrence of meiotic restitution. Cytological studies revealed adjacent nuclei distribution and incomplete cytokinesis at late meiosis II in meiocytes within CCM flowers. Immunolocalization of α-tubulin and the microtubule-associated protein MAP65-3 showed that the orientation of spindles at metaphase II and the organization of radial microtubule arrays at the tetrad stage are interfered, which explains the production of meiotically-restituted microspores. Moreover, CCM flowers showed gradually impaired expression of Aborted Microspores (AMS), a key transcription factor regulating tapetum development and meiotic cytokinesis. Interestingly, increased sucrose supply in the culture medium promoted AMS expression and partially rescued haploid microspore formation in CCM flowers. Taken together, this study suggests a role of sucrose in facilitating meiotic cytokinesis and gametophytic ploidy stability in plants.

Keywords: Meiosis; unreduced microspore; meiotic restitution; cytokinesis; microtubule; sucrose; tapetum

Introduction

Meiosis is a specialized type of cell division in which chromosomal DNA is replicated once followed by two successive nuclear divisions. Meiosis leads to genetic diversity through recombination of homologous chromosomes and production of gametes with halved chromosome numbers, which are required for genome stability across generations (Zickler and Kleckner, 2023). Most angiosperms have experienced whole-genome duplication (WGD) or polyploidization (WGP) during their evolutionary history, which plays an important role in speciation and environmental adaptation (Otto, 2007; Ren et al., 2018; Van de Peer et al., 2020). Formation of unreduced gametes through meiotic restitution, a phenomenon that defines non-reductional meiosis events, is considered the main route to WGD in flowering plants (Ramsey and Schemske, 1998).

In flowering plants, defects occurring in multiple meiosis processes can lead to formation of unreduced gametes. In Arabidopsis, potato, and horticultural plant species, dysfunction or down-regulation of the spindle regulators JASON or Parallel Spindle 1 (PS1) causes parallel and/or triad-like configurations of spindles during meiosis II, leading to failure in nuclear separation and thus production of diploid gametes and polyploid offspring (Andreuzza et al., 2015; Clot et al., 2024; d'Erfurth et al., 2008; De Storme and Geelen, 2011; Peloquin et al., 1999; Zhou et al., 2022b). Omission of the meiotic cell cycle triggered by functional defects in the meiotic cell cycle regulators Omission of Second Division 1 (OSD1) and Tardy Asynchronous Meiosis (TAM/CYCA1;2) (d'Erfurth et al., 2010; d'Erfurth et al., 2009; Pang et al., 2025; Zhou et al., 2022b) induces meiotically-restituted dyads and thus diploid gametes in multiple species. Moreover, irregular cytokinesis interferes with chromosome distribution and can also induce unreduced gamete formation (Liu et al., 2021a; Spielman et al., 1997; Takahashi et al., 2010; Yang et al., 2003; Zeng et al., 2011). In addition to genetic lesions, multiple abiotic stresses, including extreme temperatures and ultraviolet radiation, have been reported to induce meiotic restitution in plants (De Storme and Geelen, 2020; Fu et al., 2024; Mai et al., 2019; Zhou et al., 2022a).

Live-imaging microscopy technology has been increasingly used in meiosis studies in plants because of its advantage in obtaining insights into dynamic cellular processes (Prusicki et al., 2021). In this system, the dynamic behaviors of meiotic chromosomes and proteins are monitored in live meiocytes wrapped in anthers of inflorescences ex vivo cultivated in established culture medium (Prusicki et al., 2019). Based on differences in tissues, experimental purposes, and species, different types of culture medium are used for live-imaging analysis of meiosis, in which Murashige and Skoog (MS) salt has been used as the basal component with additives for suitable applications on different microscopic systems (Prusicki et al., 2021). In currently established culture systems, meiosis features including duration of meiosis and recombination rate recorded via live-imaging technology are comparable to findings obtained from fixed tissues, even under stressful environmental conditions (De Jaeger-Braet et al., 2022; France et al., 2021; Ning et al., 2021; Prusicki et al., 2019; Yang et al., 2022). Additionally, such an ex vivo system provides a convenient and reliable strategy for analyzing features of meiosis exposed to chemicals for biochemical and genetic evidence (Yuan et al., 2025). However, the potential impact of cultivating inflorescences in basal culture medium without abundant additives, such as vitamins, on meiosis has not been determined, which is relevant for developing reliable culture systems for different experimental purposes and in different plant species.

In this study, we report that Arabidopsis (Arabidopsis thaliana) flowers cultivated in a basal culture medium produce unreduced microspores due to mis-organization of the microtubule cytoskeleton during meiosis II and consequent restituted meiotic cell division. Moreover, we showed that increased sucrose supply partially rescues tapetum development and formation of haploid microspores. Taken together, this study suggests a positive role of sucrose in facilitating meiotic cytokinesis and the stability of gametophytic ploidy in Arabidopsis.

Results

Arabidopsis flowers cultivated in culture medium produce diploid microspores

To explore the effect of cultivating inflorescences in culture medium without abundant additives on meiosis in plants, we cut inflorescences from young flowering Arabidopsis (Arabidopsis thaliana) and cultivated them in a culture medium composed of half-strength Murashige and Skoog basal salt, MES (0.05% [w/v]), myo-inositol (0.01% [w/v]), sucrose (1% [w/v]), and agar (0.8% [w/v]). Flowers from inflorescences cut directly from intact plants were used as control samples. The Arabidopsis quartet 1 (qrt1) mutant, in which released microspores maintain a tetrad configuration (Francis et al., 2006), was used for analysis of meiosis products. In control flowers, only normal tetrads consisting of four haploid microspores indicating normal meiosis were observed (Fig. 1A [FIGURE:1], B and E). In flowers cultivated in culture medium (CCM) for 40–48 h, we found about 2.4% of pollen mother cells (PMCs) at the unicellular microspore stage exhibiting triad configurations, which harbored one diploid nucleus and two haploid nuclei, or a dyad containing two equally-sized microspores each showing two nuclei (Fig. 1C–E). These figures suggested that PMCs in Arabidopsis CCM flowers produce unreduced microspores likely resulting from meiotic restitution.

Fig. 1. Arabidopsis flowers cultivated in culture medium produce diploid microspores. A, A model describing the manipulation of cultivating an Arabidopsis inflorescence in culture medium. B–D, A tetrad (B), a triad (C), and a balanced-dyad (D) at unicellular microspore stage produced by control or CCM flowers in the qrt mutant. E, Graph showing the rate of unreduced microspores yielded by control and CCM flowers. The significance level was determined based on an unpaired t-test; the average rate of unreduced microspores and the number of analyzed inflorescences are shown; *** indicates P < 0.001; CCM, cultivated in culture medium; MSP, microspore. Scale bar, 10 μm.

PMCs in CCM flowers show defects in meiotic cytokinesis

To confirm the occurrence of meiotic restitution in PMCs in Arabidopsis CCM flowers, we stained PMCs at the tetrad and microspore stages in the qrt mutant with 4′,6-diamidino-2-phenylindole (DAPI). Tetrad-stage PMCs from control flowers showed separated haploid nuclei in cell corners, between which organelle bands were visible (Fig. 2A [FIGURE:2]). These tetrads developed into unicellular microspores each harboring a single haploid nucleus (Fig. 2C). In CCM flowers, adjacent distribution of two nuclei was visualized at the tetrad stage, with no organelle band between the adjacent nuclei, showing a triad configuration (Fig. 2B). These triads resulted in production of microspores with two similarly-sized nuclei indicating diploid microspores (Fig. 2D and E). At later developmental stages, triad microspores showed sticky cell walls that blocked DAPI entry into cells with failed nuclear staining (Fig. 2F).

The irregular distribution of haploid nuclei suggested that cell walls were not successfully built, indicating a defect in cytokinesis. To investigate this, we stained tetrad-stage PMCs with DAPI and aniline blue, which labels callose, the main component of meiotic cell walls during meiosis and early microspore stages. In a tetrad, callosic cell walls were constructed between four isolated nuclei, preventing them from fusing and thus facilitating formation of haploid microspores (Fig. 2G). Remarkably, tetrad-stage PMCs in CCM flowers exhibited failed and/or incomplete assembly of callosic cell walls, leading to adjacent localization of nuclei (Fig. 2H–L). These observations revealed a defect in meiotic cytokinesis in PMCs from CCM flowers.

Figure 2. Pollen mother cells in flowers cultivated in culture medium show defects in meiotic cytokinesis. A and B, DAPI-staining of PMCs at the tetrad stage showing normal (A) and adjacent (B) nuclei distribution. C–F, DAPI-stained unicellular microspores observed in control (C) showing normal tetrad configuration and flowers cultivated in culture medium (CCM) (D–F) showing triad configuration. G–L, Combined DAPI and aniline blue staining of tetrad-stage PMCs in control (G) and CCM flowers (H–L). Scale bars, 10 μm.

PMCs in CCM flowers show irregular chromosome distribution at the end of meiosis II

To further characterize meiotic defects in Arabidopsis CCM flowers, we analyzed chromosome spreads by DAPI staining. In control flowers, meiocytes at pachytene stage displayed full juxtaposition of homologous chromosomes (homologs) (Fig. 3A [FIGURE:3]), indicating complete homolog pairing and synapsis. Five bivalents were consistently observed at diakinesis and metaphase I (M I) (Fig. 3B and C), manifesting completion of meiotic recombination. Homologs separated at anaphase I (A I) and temporarily decondensed at interkinesis, which recondensed and aligned at two cell poles at M II by pulling force from two spindles (Fig. 3D–F). At telophase II (T II), separation of sister chromatids led to formation of four haploid chromosome sets, each developing into a haploid nucleus at the tetrad stage (Fig. 3G and H). In CCM flowers, meiocytes during meiosis I did not show obvious defects (Fig. 3I–M), indicating regular meiotic recombination and homolog separation. However, defective orientation of aligned chromosomes at M II was visualized (Fig. 3N and O), implying improper organization and/or mis-orientation of spindles. At T II, adjacent localization of nuclei indicative of irregular chromosome distribution was observed (Fig. 3P and Q), leading to production of tetrad meiocytes showing triad configuration (Fig. 3R–T). Hence, the observed meiotically-restituted unreduced microspores likely resulted from defective distribution of chromosome sets and nuclei at the end of meiosis II.

Fig. 3. PMCs in flowers cultivated in culture medium show adjacent nuclei distribution at the end of meiosis II. A–T, DAPI-stained meiotic chromosome spreads at pachytene (A and I), diakinesis (B and J), metaphase I (C and K), anaphase I (D and L), interkinesis (E and M), metaphase II (F, N and O), telophase II (G, P and Q), and tetrad (H and R–T) stages in control flowers (A–H) and flowers cultivated in culture medium (I–T). Scale bars, 10 μm.

PMCs in CCM flowers show irregular spindle and phragmoplast organization at meiosis II

To determine whether lesions in meiotic chromosome distribution and cytokinesis in CCM flowers are induced by defects in microtubular cytoskeleton, we analyzed organization of spindles and phragmoplasts by performing immunolocalization of tubulin and the microtubule-associated protein MAP65-3 using anti-α-tubulin and anti-GFP antibodies in the pMAP65-3::MAP65-3-GFP reporter line (Sofroni et al., 2020). In meiocytes from both control and CCM flowers, a spindle was built at M I with MAP65-3 protein co-localizing with microtubules (Fig. 4A [FIGURE:4]). At A I and interkinesis, a phragmoplast structure was organized between separated homologs, and MAP65-3 localized at the middle region of the phragmoplast (Fig. 4B and C). These data confirmed that CCM flowers have no defect in meiosis I. In control meiocytes at M II, two spindles were perpendicularly organized to drive segregation of sister chromatids at T II, and MAP65-3 displayed a similar localization pattern as in meiosis I (Fig. 4D and E). At the tetrad stage, mini-phragmoplasts composed of radial microtubule arrays (RMAs) were assembled between isolated haploid nuclei, and MAP65-3 localized at the center of mini-phragmoplasts (Fig. 4F). In CCM flowers, the localization pattern of MAP65-3 was not altered (Fig. 4G–P). However, meiocytes at M II and T II stages displayed triangle configurations of spindles and phragmoplasts (Fig. 4H–I), indicating irregular orientation. At the tetrad stage, triads showing adjacent localization of nuclei and omission of RMAs were visualized (Fig. 4J–N). Moreover, in some tetrads showing separated haploid nuclei, RMAs and MAP65-3 were not regularly assembled between nuclei (Fig. 4O and P). The defective microtubule assembly and/or organization could explain the lesions in meiotic chromosome distribution and cytokinesis in CCM flowers.

Fig. 4. Meiocytes in flowers cultivated in culture medium show irregular microtubule organization during meiosis II. A–P, Immunolocalization of alpha-tubulin (green) and MAP65-3-GFP (red) in meiocytes at metaphase I (A), anaphase I (B), interkinesis (C), metaphase II (D and G), telophase II (E, H and I), and tetrad (F, J–P) stages showing normal (A–F) or abnormal organization and/or assembly (G–P) observed in control flowers and flowers cultivated in culture medium. Scale bar, 10 μm.

Increased sucrose supply partially rescues AMS expression in CCM flowers

It has been reported that normal development of tapetum is needed for faithful organization of RMAs and meiotic cytokinesis in Arabidopsis (Tidy et al., 2022). The lesions in meiotic cell wall formation and RMA organization in meiocytes led us to hypothesize that meiotic restitution in CCM flowers is triggered by attenuated function and/or development of the tapetum. To test this, we analyzed expression of Aborted Microspores (AMS), a transcription factor required for tapetum development, dysfunction of which induces defective meiotic cytokinesis and thus meiotic restitution, by performing live-imaging using CCM flowers from a pAMS::AMS-GFP reporter (Xiong et al., 2016). In control flowers, anthers at the tetrad and unicellular microspore stages showed expression of AMS-GFP specifically in the tapetal cell layer (Fig. 5A [FIGURE:5] and B). In tetrad-stage anthers at one-day post cultivation (1 dpc) in the medium, no obvious alteration was detected in AMS-GFP expression (Fig. 5C). However, reduced AMS-GFP expression was visualized in anthers at 2 dpc, and no AMS-GFP was detected at 3 dpc (Fig. 5E and G). These data indicated that AMS expression is damaged in CCM flowers.

It has been reported that faithful tapetum development relies on normal sugar metabolism in flowers (Borghi, 2025; Liu et al., 2021b). We wondered whether damaged AMS expression in CCM flowers is due to shortage of sucrose supply in anthers. Therefore, we monitored AMS expression in CCM flowers exposed to elevated sucrose concentration. In most flowers from 1 to 3 dpc, we observed normal AMS-GFP expression in anthers at the tetrad stage, indicating that increased sucrose supply partially rescued AMS expression in the tapetum (Fig. 5I, K and M). Because sucrose breaks down into other sugars to induce downstream cellular responses (Yoon et al., 2021), we tested whether fructose, a metabolite of sucrose, has a similar effect on AMS expression in CCM flowers. CCM flowers exposed to 1% fructose showed normal AMS expression at 1 dpc, but exhibited reduced and impaired AMS expression at 2 and 3 dpc, respectively (Fig. 5D, F and H). Under 10% fructose condition, AMS expression in most flowers at 2 and 3 dpc was recovered (Fig. 5J, L and N). These findings reveal a positive impact of sucrose and its metabolite on AMS expression in anthers.

Fig. 5. Increased sucrose or fructose supply rescues AMS expression in the tapetum. A and B, Expression of AMS-GFP in the tapetum of anthers at the tetrad (A) and microspore (B) stages in control flowers. C–H, Expression of AMS-GFP in the tapetum of anthers at the tetrad stage in flowers cultivated in culture medium containing 1% sucrose (C, E and G) or 1% fructose (D, F and H) for 24 (C and D), 48 (E and F) and 72 h (G and H). I–N, Expression of AMS-GFP in the tapetum of anthers at the tetrad stage in flowers cultivated in culture medium containing 10% sucrose (I, K and M) and 10% fructose (J, L and N) for 24 (I and J), 48 (K and L) and 72 h (M and N). Scale bar, 50 μm.

Increased sucrose supply partially rescues haploid microspore formation

To test whether increased sucrose supply could complement meiotic cytokinesis defects in CCM flowers, we quantified rates of unreduced microspores in CCM flowers exposed to different sucrose concentrations. In control flowers, only tetrads and haploid microspores were observed (Fig. 6A [FIGURE:6], E and I). In flowers cultivated in culture medium with 1% sucrose, meiocytes showing meiotic restitution at the tetrad stage were visualized and about 3.1% unreduced microspores were recorded (Fig. 6B–D, F–H and I). A similar rate of unreduced microspores was found in flowers grown in medium without sucrose as in 1% sucrose-supplied medium (Fig. 6I). Interestingly, flowers cultivated in medium with 10% sucrose yielded a significantly lower rate (~1.3%) of unreduced microspores (Fig. 6I, P < 0.01), suggesting partially rescued meiotic cytokinesis and haploid microspore formation.

Fig. 6. Increased sucrose supply in culture medium partially reduces diploid microspore formation. A–H, Orcein-staining of meiocytes at tetrad stage (A–D) and unicellular microspores (E–H) in control flowers (A and E) and flowers cultivated in culture medium (B–D, F–H) showing tetrad (A and E), triad (B and F), balanced-dyad (C and G) or unbalanced-dyad (H) configuration. I, Graph showing the rate of diploid microspores yielded by control flowers and flowers in culture medium containing 0%, 1% and 10% sucrose, respectively. Significance levels were determined based on unpaired t-tests; the average rate of unreduced microspores and the number of inflorescences are shown; *** indicates P < 0.001; ** indicates P < 0.01; ns indicates P > 0.05; MSP, microspore. Scale bar, 10 μm.

Discussion

In this study, we showed that Arabidopsis flowers cultivated in culture medium produce unreduced microspores due to occurrence of meiotic restitution caused by defective meiotic cytokinesis. Further cytological studies revealed that the orientation of spindles and phragmoplasts during meiosis II is altered, resulting in irregular formation and/or organization of RMAs at the tetrad stage in CCM PMCs. The altered orientation of spindles and phragmoplast at meiosis II in CCM flowers is possibly due to interfered expression and/or function of the spindle regulator JASON and/or AtPS1 (De Storme and Geelen, 2011), which have been proposed to mediate the response of microtubule organization to environmental stimulus (Cabout et al., 2017; Fu et al., 2024). However, tetrads showing normal nuclei distribution but failed assembly of RMAs and MAP65-3 localization between separated nuclei (Fig. 4P) suggest that formation and/or composition of organelle bands, which act as physical barriers between metaphase II spindles and thus ensure correct chromosome distribution (Brownfield et al., 2015), may be attenuated independently of impacted JASON function (Gasser et al., 1988; Koç and De Storme, 2022).

The occurrence of meiotic restitution and microtubule organization defects have not been reported in previous studies that cultured Arabidopsis inflorescences in growth medium for live-imaging microscopy (Prusicki et al., 2019; Sofroni et al., 2020; Valuchova et al., 2022; Wijnker et al., 2019; Yang et al., 2020; Yang et al., 2022; Yuan et al., 2025). The culture medium used in this study only contained myo-inositol without other vitamins, such as nicotinic acid, pyridoxine hydrochloride, glycine, and thiamine hydrochloride, which play important roles in metabolic regulation including energy metabolism, amino acid synthesis, oxidation-reduction reactions, stress response, and cell development in plants (Berglund et al., 2017; Schnellbaecher et al., 2019; Sultana et al., 2019). Thus, we speculate that the meiosis defects we observed are induced by cellular lesions due to lack of chemicals necessary for meiocyte development (Prusicki et al., 2021). Hence, the components and possibly their dosages in the culture medium should be tested and delicately modified for establishment of an ex vivo culture system for plant meiosis study. On the other hand, the average frequency of unreduced microspores in CCM flowers is quite low (less than 3%), which plausibly makes it difficult to capture lesions under fast-moving and dynamic intracellular conditions. Considering the complexity of chromosome dynamics during meiosis, especially during recombination, minor differences may exist between features of meiosis captured in flowers grown in the ex vivo culture system and those under natural conditions.

It has been reported that normal development of tapetum at early flower stages is needed for successful RMA organization and assembly and regular configuration of cytokinetic cell walls during meiosis in Arabidopsis (Liu et al., 2017; Tidy et al., 2022). The defects in callosic cell walls together with impairment of AMS expression in anthers suggest that meiotic restitution in CCM flowers could be caused by damaged development and function of the tapetum, which thereafter attenuates expression of meiosis genes or metabolism of enzymes and components for organelle band formation and cell wall assembly in meiocytes (Biswas and Chaudhuri, 2024; Lei and Liu, 2020; Muro et al., 2025; Wei and Ma, 2023). Whether and how the tapetum plays a role in regulating expression and/or function of JASON and AtPS1, or other microtubule regulators, awaits further study. We found that increased sucrose concentration in the culture medium promoted AMS expression and haploid microspore formation, which could be attributed to compensated energy supply or activation of signaling pathways mediated by sucrose and/or its metabolites in anthers (Borghi, 2025; Liu et al., 2021b; Wang et al., 2022; Yoon et al., 2021).

It can be noted that the rate of unreduced microspores largely varies between individual CCM inflorescences (Fig. 6I), which may result from differences in inherent sucrose abundance in those inflorescences. In multiple plant species, regulatory modules of tapetum development and meiosis progression are tightly associated with factors in sugar metabolism and/or signaling (Lei and Liu, 2020; Liu et al., 2021b; Sun et al., 2025; Wang et al., 2023). In addition, in rice, TDR Interacting Protein 2 (TIP2), a basic helix-loop-helix protein required for tapetum development, regulates expression of carbohydrate-active glycosyltransferases and glycosyl hydrolases in the tapetum, dysfunction of which has been found to cause arrested meiosis progression (Fu et al., 2014; Wang et al., 2025). These findings suggest that the tapetum may influence fidelity of meiotic cytokinesis by regulating sugar metabolism. We propose that partially rescued microsporogenesis by increased sucrose supply is a consequence of recovered tapetum function.

Overall, our study reveals a role of sucrose in facilitating tapetum development and meiotic cytokinesis. Moreover, cultivating flowers in medium with modified components including sugars for ensuring anther development could potentially be developed as a strategy for inducing unreduced gametes in polyploid breeding programs.

Material and Methods

Plant materials and growth conditions

Arabidopsis thaliana mutant quartet (qrt) (Francis et al., 2006), the pMAP65-3::GFP-MAP65-3 (Sofroni et al., 2020) and pAMS::AMS-GFP (Xiong et al., 2016) reporters were used in this study. Seeds were germinated in soil for 6–8 days and seedlings were transferred to soil and cultivated in growth chambers with 16 h day/8 h night, 20°C, and 50% humidity. For flower cultivation, young inflorescences were cut with tips embedded into the medium. A suitable amount of distilled water was added to the medium to avoid drying of inflorescences.

Preparation of culture medium

The culture medium used in this study was prepared by dissolving basal MS salt (0.5×), MES (0.05% [w/v]), myo-inositol (0.01% [w/v]), sucrose (1% [w/v]), and agar (0.8% [w/v]) in distilled water with pH adjusted to 5.7.

Cytological analysis of meiocytes and microspores

Orcein staining, 4,6-diamidino-2-phenylindole (DAPI) staining, and aniline blue staining of meiocytes were performed as described previously (Fu et al., 2024). Quantification of microspores was performed at 48 h post cultivation of flowers in the medium.

Preparation of chromosome spreads

Inflorescences of young Arabidopsis were fixed in precooled Carnoy's fixative for at least 24 h. Meiosis-staged flower buds were washed twice with distilled water and once with citrate buffer (10 mM, pH = 4.5), followed by incubation in digestion enzyme mixture (0.3% pectolyase and 0.3% cellulase in citrate buffer, 10 mM, pH = 4.5) at 37°C for 2.5 h. Subsequently, digested flower buds were washed once in distilled water and macerated in distilled water on a glass slide. Two aliquots of 60% acetic acid were added to the slide, which was dried on a hotplate at 45°C for 2 min. The slide was washed with ice-cold Carnoy's fixative and then air-dried. DAPI (5 μg/mL) diluted in antifade mounting medium was added to the slide, and the coverslip was mounted and sealed with nail polish.

Immunolocalization of microtubules and MAP65-3 protein

Immunolocalization assays were performed following Liu et al. (2017) and Wang et al. (2014). Antibodies against α-tubulin (Lei et al., 2020) and GFP (Zhao et al., 2023) were diluted 1:500 and 1:300, respectively. Secondary antibodies have been described previously (Lei et al., 2020; Zhao et al., 2023).

Live-imaging of reporters

To analyze AMS expression, anthers in flowering pAMS::AMS-GFP reporter were isolated and placed on a glass slide with a drop of distilled water added to samples, which was then mounted with a coverslip and examined under an inverted fluorescence microscope. Developmental stages of anthers were determined by checking released PMCs or microspores in the anthers.

Microscopy

Fluorescence microscopy was performed using an Olympus IX83 inverted fluorescence microscope equipped with an X-Cite lamp and a Prime BSI camera. Bifluorescent images and Z-stacks were processed using ImageJ.

Statistical analysis

Significance analysis was performed using unpaired t-test with GRAPHPAD PRISM (v.8), and significance level was set as P < 0.05. Variation bars indicate SD.

Funding

This study was supported by Hubei Provincial Natural Science Foundation of China (2024AFB695), the Fundamental Research Funds for the Central Universities, South-Central Minzu University (CZZ24011), Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding (2021C02071–6 to Z.R.), Zhejiang Provincial Natural Science Foundation (ZCLTGN24C1601 to Z.R.), and Zhejiang Sci-Tech University Start-up Fund (22052138-Y to Z.R.).

Data availability statement

The data that support the findings of this study are available from the corresponding author (B.L.) upon reasonable request.

CRediT authorship contribution statement

H.F., Y.C., X.C., H.H. and J.W. contributed to investigation; C.W. and Z.R. contributed to data analysis; B.L. conceived project, analyzed data, wrote, and edited the manuscript. All authors have read and agreed with the manuscript prior to submission.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.

Acknowledgements

The authors thank Arp Schnittger (Universität Hamburg) for sharing the pMAP65-3::MAP65-3-GFP reporter; they also thank Zhongnan Yang (SJTU) and Yue Lou (SHNU) for sharing the pAMS::AMS-GFP reporter.

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