Effects of fertilization of male gametes with heavy-ion beam irradiation on embryo and endosperm development in Cyrtanthus mackenii

Abstract

Heavy-ion beams are widely used for mutagenesis. The type and size of the induced mutations vary depending on the ion species and velocity. DNA damage response of male gametes during pollen tube growth has been investigated using heavy-ion beam irradiation to the pollen of Cyrtanths mackerii (Amaryllidaceae), indicating that DNA damage induced by argon-ion beam is more difficult to repair than that by carbon-ion beam. In this study, we investigated the effects of DNA damage or mutations in the male gametes with argon-ion irradiation on double fertilization and subsequent embryo and endosperm development and compared the results with carbon-ion irradiation. In immature seeds after pollination with argon-ion-irradiated pollen grains at 10 Gy, there were two types of embryo sacs with embryo and endosperm and with egg cell/zygote and endosperm. The proportion of embryo sacs in the latter type was higher when using argon-ion-irradiated pollen than carbon-ion-irradiated pollen at 40 Gy, suggesting that qualitative differences between the two kinds of irradiation influenced embryo development. In many endosperms after pollination with the irradiated pollen, abnormal chromosome separation and enlarged endosperm nuclei were observed. This indicated that the chromosomal abnormalities in the irradiated male gametes were transmitted to the endosperm nuclei. The enlarged nucleus formation was higher in pollen irradiated with an argon-ion beam than in that with a carbon-ion beam. In conclusion, argon-ion beams, even at low doses, induce distinctive development of embryo and endosperm, making them valuable for investigating double fertilization.

The heavy-ion beam is a type of ionizing radiation and is composed of accelerated ions heavier than helium. Thus far, mutation-inducing techniques using heavy-ion beams have been developed mainly in plants (Tanaka et al. 2010; Kazama et al. 2018; Abe et al. 2021; Hirano et al. 2022). One of the characteristics of heavy-ion beams is their high linear energy transfer (LET, keV µm⁻¹), which is a unit of energy deposited per unit length along the pass of the radiation through a substance. X-rays and γ-rays are classified as low-LET radiation because the values corresponding to LET are several keV µm⁻¹ or less. In contrast, heavy-ion beams are classified as high-LET radiation, with LET values varying depending on the ion species and their velocity. LET of heavy-ion beams used for biological irradiation in RIKEN RI beam factory (RIBF) range from 23 to 4,000 keV µm⁻¹ (Ryuto et al. 2008). The heavy-ion beams with high LET values form high-density ionization regions along their path. When cells are irradiated with heavy-ion beams, localized DNA damage, which is difficult to repair, is induced (Pastwa et al. 2003; Sage and Harrison 2011).

Various plant species have been irradiated with carbon, nitrogen, neon, argon, and iron ions for mutation breeding and functional studies. The results of those studies have revealed that the type and size of the induced mutations depend on the LET values. For example, in Arabidopsis thaliana, carbon-ion beams at 23 and 30 keV µm⁻¹ frequently induce small deletions of a few bases, while an argon-ion beam at 290 keV µm⁻¹ induces large deletions and chromosomal rearrangements, such as inversions and translocations, at high frequency (Kazama et al. 2011, 2017; Hirano et al. 2012, 2015). LET has also been reported to affect mutation induction in rice (Ichida et al. 2019; Abe et al. 2021; Morita et al. 2021).

Heavy-ion beam irradiation of pollen has been used as a genetic research tool and to obtain mutants in dioecious plants (Naito et al. 2005; Kazama et al. 2016). Moreover, it serves as an experimental system to observe the first cell division process after irradiation (Hirano et al. 2022). In Cyrtanthus mackenii, DNA damage responses in male gametes during pollen tube growth have been investigated using bicellular pollen exposed to carbon- or argon-ion beams (Hirano et al. 2013, 2021). In the irradiated generative cells, sperm cell formation decreased with the increase in absorbed dose, and lagging chromosomes and chromosomal bridges were observed. It has been shown that the DNA damage in the male gametes with argon-ion beam irradiation remains unrepaired for a longer time than that with carbon-ion beam irradiation (Hirano et al. 2021), indicating that the DNA damage induced by argon-ion beam is difficult to repair in the male gametes. The developmental processes of embryos and endosperm after pollination with pollen grains irradiated with a carbon-ion beam have also been reported. Two types of embryo sacs with an embryo and endosperm and with an egg cell or an undivided zygote and endosperm were observed (Hirano et al. 2024). Furthermore, the embryos and endosperm showed various DNA contents. Although pollen irradiated with X-rays or γ-rays has long been used for haploid induction (Sestili and Ficcadenti 1996), little is known about the effects of DNA damage or mutations in male gametes on double fertilization and subsequent embryo and endosperm development.

In this study, the behavior of male gametes during double fertilization was investigated by crossing C. mackenii pollen irradiated with an argon-ion beam and observing embryo and endosperm development. By comparing the developmental process under carbon-ion beam irradiation, we aimed to determine whether qualitative differences between the two kinds of irradiation affect embryo and endosperm development. In addition, to characterize the endosperm development derived from pollination with carbon- and argon-ion beam-irradiated pollen, the localization of actin filaments in the embryo sacs was examined.

Materials and methods

Plant materials and fixation

Cyrtanthus mackenii Hook. f. (Amaryllidaceae) was grown in fields at the University of Miyazaki, Miyazaki, Japan. Mature pollen grains with the anthers were collected and dried in 0.2 mL tubes at room temperature for 1 day and then stored at −20°C with a silica gel. Heavy-ion beam irradiation of the pollen grains was performed at the RIKEN RIBF. After irradiation with an argon-ion beam (280 keV µm⁻¹) at doses of 10 Gy and 40 Gy and a carbon-ion beam (22.5 keV µm⁻¹) at doses of 10 Gy and 40 Gy, the pollen grains were stored at −20°C. Two days after emasculation, the pollen grains were pollinated to the stigma. At 2 or 14 days after pollination (DAP), the pistils were fixed in FAA (formaldehyde: acetic acid: ethanol) or 4% paraformaldehyde in phosphate-buffered saline (PFA) for at least 24 h. They were then placed in 70% ethanol and stored at 4°C.

Observation of pollen tube growth and embryo sacs

At 2 DAP, the FAA-fixed pistils were hydrolyzed in 1 M NaOH at 60°C for 20 min and then washed thrice with distilled water. The pistils were stained with 0.2% aniline blue overnight. Pollen tubes in the pistil were then observed under an optical microscope (BX51-34-FL-2; Olympus).

Paraffin sectioning was performed on the immature seeds fixed in FAA at 14 DAP. The samples were sectioned to a thickness of 10 µm using a microtome and stained with hematoxylin. Serial sections were photographed using the optical microscope. To calculate the standard nucleus size of the embryo and endosperm using the sections, the nucleus area in the embryo and endosperm of three embryo sacs obtained after pollination of unirradiated pollen was measured using ImageJ (Schneider et al. 2012). The nuclei with nucleoli were used for the measurement. The nucleus area in each endosperm was measured for analysis of enlarged endosperm nucleus formation.

Embryo sac isolation and actin filament visualization

Embryo sacs were isolated from the immature seeds fixed in PFA at 14 DAP under a stereomicroscope (SZ2-ILST; OLYMPUS). The isolated embryo sacs were transferred onto a polylysine-coated coverslip and immersed in microtubule-stabilizing buffer [50 mM 1,4-piperazinediethanesulfonic acid, 5 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1 mM MgCl₂, and 2% (v/v) glycerol, pH 6.9] for 10 min. The embryo sacs were treated with PBS containing 0.05% Tween 20 for 5 min and a blocking agent (Image-iT FX Signal Enhancer; Thermo Fisher Scientific) for 60 min. The embryo sacs were then stained with 10 units mL⁻¹ Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific) in PBS for 2 h and 1 µg mL⁻¹ Hoechst 33258 in PBS for 10 min. After washing thrice with PBS, the embryo sacs were mounted on coverslips in an antifade reagent (SlowFade Gold Antifade reagent, Thermo Fisher Scientific). Images were obtained at 1.0 µm steps along the Z-axis using a confocal laser-scanning microscope (LSM 700; Carl Zeiss). Z-stack images of a whole embryo sac were acquired to confirm the development of the embryo and endosperm.

Results

Pollen germination and pollen tube growth after pollination with argon-ion beam-irradiated pollen grains

To determine the effects of argon-ion beam irradiation on pollen germination and pollen tube growth, anillin blue staining was performed at 2 DAP. For both unirradiated and 40 Gy-irradiated pollen, many pollen grains germinated on the stigma (Fig. 1A, B), and the pollen tubes elongated in the styles and reached the ovules (Fig. 1C–F). The pollen grains irradiated with the argon-ion beam at 0 (control), 10, and 40 Gy were pollinated to 72, 105, and 91 pistils, respectively, and the number of successfully collected ovaries at 14 DAP were 67 (93%) at 0 Gy, 98 (93%) at 10 Gy, and 73 (80%) at 40 Gy.

Fig. 1. Pollen germination and pollen tube growth in the pistils.

The pistils were fixed at 2 days after pollination (DAP) with unirradiated pollen grains (A, C, E) or argon-ion beam-irradiated pollen grains at 40 Gy (B, D, F), and the pollen tubes were stained with aniline blue. (A, B) The pollen grains germinated on the stigma. (C, D) The pollen tubes elongated in the style and (E, F) penetrated the ovules. The arrows and arrowheads indicate the pollen tubes and micropyle regions in the ovules, respectively. Scale bars=100 µm.

Embryo and endosperm development after pollination with argon-ion beam-irradiated male gametes

Histological observation of the ovaries at 14 DAP was conducted to analyze embryo and endosperm development. After pollination with unirradiated pollen, the embryo and endosperm in the syncytial phase were observed in the embryo sacs of the immature seeds, and the developmental stage of the embryos was from the 8-cell stage to the globular stage (Fig. 2A, B). Although embryos and endosperm were also observed in the embryo sacs derived from pollen grains irradiated with 10 Gy of argon-ion beam, embryo development was inhibited (1- to 4-cell stage; Fig. 2C, D). In addition, embryo sacs with an egg cell or an undivided zygote and endosperm were observed, and some of the embryo sacs derived from pollination with irradiated pollen grains showed a narrowed structure (Fig. 2E, F). In the embryo sacs derived from pollen grains irradiated with 40 Gy of argon-ion beam, only one normal type with a globular-stage embryo and endosperm were observed, while all others were unfertilized. We categorized the embryo sacs into three groups based on embryo and endosperm development (Table 1): embryo sacs with an embryo and endosperm (embryo+/endosperm+), with an egg cell or an undivided zygote and endosperm (embryo−/endosperm+), and with an embryo and a central cell with a secondary nucleus (embryo+/endosperm−). All embryo sacs formed embryos and endosperm in the unirradiated condition. In contrast, at 10 Gy irradiation, only 15% (5/33) of embryo sacs formed embryos and endosperm, and 85% (28/33) of embryo sacs formed an egg cell/zygote and endosperm. There were no immature seeds without endosperm development.

Fig. 2. Embryo and endosperm development at 14 DAP.

(A, C, E) Whole images of embryo sacs and (B, D, F) their respective magnified images of the embryos, indicated by the area enclosed by the dotted line in the embryo sac images. (A, B) The embryo sacs with embryo and endosperm were formed in the immature seeds derived from unirradiated and (C, D) 10 Gy argon-ion-irradiated pollen grains. (E, F) After pollination with irradiated pollen grains, embryo sacs with an egg cell or zygote and endosperm were observed. The arrows and arrowheads indicate the embryo (or an egg cell or zygote) and endosperm nuclei, respectively. Scale bars=100 µm.

　

Table 1. Embryo sac types developed after pollination with heavy-ion beam-irradiated pollen.

Irradiation		No. of embryo sac type (%)			Total
	Dose (Gy)	embryo+/endosperm+	embryo−/endosperm+	embryo+/endosperm−
	0	60 (100)	0 (0)	0 (0)	60
Argon ion (280 keV µm−1)	10	5 (15)	28 (85)	0 (0)	33
	40	1 (100)	0 (0)	0 (0)	1
Carbon ion (22.5 keV µm−1, Hirano et al. 2024)	10	57 (98)	1 (2)	0 (0)	58
	40	66 (86)	11 (14)	0 (0)	77

In endosperm derived from pollination with the irradiated pollen, numerous abnormal chromosome segregation and nuclear division abnormalities were observed, regardless of the presence or absence of an embryo; nuclei with chromosomal bridges (Fig. 3A) or with lagging or exposed chromosomes (Fig. 3B), two nuclei connected without division (Fig. 3C), and enlarged nuclei (Fig. 3D). To investigate the frequency of the enlarged endosperm nucleus formation, the nucleus size was measured. The sizes of embryo and endosperm nuclei derived from unirradiated pollen were 95.2±7.4 (S.D.) µm² and 142.8±5.7 (S.D.) µm², respectively. The enlarged nuclei were defined as those larger than four times the average embryo nucleus size (380.8 µm²), and the nuclei up to about 15 times the size (1,470.4 µm²) were observed. The enlarged endosperm nuclei were also observed after pollination with unirradiated pollen (Table 2). The formation rate increased after pollination with heavy-ion beam-irradiated pollen, especially 25.6% of endosperms derived from 10 Gy argon-ion beam-irradiated pollen formed the enlarged endosperm nuclei (Table 2).

Fig. 3. Abnormal endosperm nuclei formed at 14 DAP after pollination with pollen irradiated with an argon-ion beam at 10 Gy.

(A) Chromosomal bridge formation, (B) chromosomal separation abnormality, (C) an undivided nucleus, and (D) an enlarged nucleus. The arrows indicate abnormal endosperm nuclei. Scale bars=50 µm.

　

Table 2. Enlarged endosperm nucleus formation in embryo sacs developed after pollination with heavy-ion beam-irradiated pollen.

		Ar ion (Gy)	Carbon ion (Gy)
	0 Gy	10	10	40
formation rate (%)	3.5	25.6	12.0	6.6

Actin filament in the embryo sac

As in the case of pollen irradiated with a carbon-ion beam (Hirano et al. 2024), pollination of argon-ion-irradiated pollen was found to induce abnormal embryo and endosperm development. To characterize these processes, the localization of actin filaments in isolated embryo sacs was examined after pollination with pollen irradiated with 40 Gy carbon-ion or 10 Gy argon-ion beams. Each dose was chosen as it was capable of abnormal embryo sac development. In the unirradiated control, actin filaments were detected as bundles along the chalazal-micropylar axis throughout the embryo sac (Fig. 4A). Accumulation was also observed in the synergid cells and at the nucleolus in the endosperm nuclei. The fluorescent signal of actin tended to be weak in most of the embryo sacs derived from heavy-ion beam irradiated pollen (Fig. 4B–E). In the embryo sacs induced by pollination with pollen irradiated with a 40 Gy carbon-ion beam, the actin filaments were not detected as clear bundles, despite accumulating in the synergid cells, and the endosperm nuclei were clustered in the center of the embryo sacs (Fig. 4B). In the embryo sacs with enlarged endosperm nuclei, the actin bundles were detected around the enlarged endosperm nuclei (Fig. 4C, D). In a certain embryo−/endosperm+ embryo sac derived from 10 Gy argon-ion irradiation, vigorous proliferation of endosperm nuclei was also observed (Fig. 4E).

Fig. 4. Actin filament distribution in embryo sacs isolated from ovules at 14 DAP.

(A) The embryo sacs derived from pollination with unirradiated pollen grains, (B, C) pollen grains irradiated with a carbon-ion beam at 40 Gy, and (D, E) pollen grains irradiated with an argon-ion beam at 10 Gy. The white and magenta arrows indicate the embryo cells and egg cell/zygote cell, respectively. The white and yellow arrowheads indicate synergid cells and the enlarged endosperm nuclei. Scale bars=100 µm.

Discussion

In this study, we investigated the effects of argon-ion beam irradiated pollen on the pollination process and early stage of embryo and endosperm development, and characteristic phenomena were observed, despite using low absorbed doses compared to previous pollen irradiation studies. In the pre-fertilization process, the pollen tubes from pollen grains irradiated with a 40 Gy argon-ion beam reached the ovules (Fig. 1), but most of the ovules were not developed. This suggested that double fertilization did not occur due to extensive DNA damage and subsequent repair defects. Cell division in the embryos and endosperm development was also thought to cease completely at an early stage due to structural aberrations of chromosomes. We analyzed immature seeds at 14 DAP to compare the effects of fertilization of argon-ion beam-irradiated male gametes on embryo and endosperm development with those of carbon-ion beam fertilization (Fig. 2, Table 1). A characteristic type of embryo sacs, embryo−/endosperm+ embryo sacs, were formed in the immature seeds derived from argon-ion- and carbon-ion-irradiated pollen grains. The proportion of embryo−/endosperm+ embryo sacs was higher after pollination with 10 Gy argon-ion-irradiated pollen (85%) than with 40 Gy carbon-ion-irradiated pollen (14%, Hirano et al. 2024). Although normal embryo sacs (embryo+/endosperm+) were also formed after pollination with pollen treated with 10 and 40 Gy of argon-ion irradiation, embryo development was arrested (1–4 cell stage). Thus, argon-ion beam irradiation in male gametes had a severe inhibitory effect on embryo development. Argon- and carbon-ion beams have different LET values and induce different types and sizes of mutations (Kazama et al. 2011, 2017; Hirano et al. 2012, 2015). Thus, qualitative differences in DNA damage and mutations in male gametes influence embryo development, and large deletions and/or chromosomal rearrangements induced by high-LET irradiation are key factors. From another perspective, embryo+/endosperm− type embryo sacs did not develop after pollination with carbon-ion- or argon-ion-irradiated pollen, suggesting that the embryo may be more strictly regulated than the endosperm by checkpoints in the cell cycle.

When C. mackenii pollen was irradiated with heavy-ion beams, unreduced sperm cells, known as generative-cell-like sperm cells (GC-like SCs) were formed. GC-like SCs are cells with generative-cell-like nuclei and sperm-cell-like microtubule arrays. They complete pollen mitosis (PM) II, but the chromosomes fail to separate (Hirano et al. 2013, 2021). The unreduced sperm cell may be one of the causes of the formation of the embryo−/endosperm+ type embryo sac by single fertilization with an egg cell or a central cell. In the case of fertilization with an egg cell, mitosis may cease under the influence of the checkpoints, and the endosperm may develop autonomously. Autonomous endosperm development has been reported in some plant species (Sestili and Ficcadenti 1996; Rojek and Ohad 2023). Moreover, it has been reported that embryogenesis is not essential for endosperm development in Arabidopsis, although the endosperm is essential for embryo development (Xiong et al. 2021). This seems to be consistent with the phenomenon we observed in Cyrtanthus.

In endosperm derived from argon-ion beam-irradiated pollen, abnormal chromosome separation at nuclear division and formation of enlarged endosperm nuclei were observed (Figs. 3 and 4). As well as the formation of GC-like SCs, nuclear division would be inhibited by multiple chromosome bridges. Subsequent repeated cell division may have led to the formation of the enlarged endosperm nuclei. Chromosomal bridges were frequently observed during PMII in generative cells of C. mackenii irradiated with heavy-ion beams (Hirano et al. 2013, 2021), suggesting that the mutations and DNA damage were transmitted to the endosperm nuclei and caused the abnormality. The enlarged nucleus formation was higher in pollen irradiated with an argon-ion beam than in that with a carbon-ion beam (Table 2). The formation of GC-like SCs was observed more frequently with C-ion irradiation than with Ar-ion irradiation at the same absorbed dose (Hirano et al. 2013, 2021). On the contrary, DNA double-strand breaks (DSBs) in the carbon-ion beam-irradiated male gametes were repaired during PMII, but those in the argon-ion beam-irradiated male gametes remained after PMII. Thus, one possibility is that the repair of DSBs after fertilization contributes to the increased formation of enlarged endosperm nuclei.

Actin filaments play an important role in coenocytic endosperm development. Actin bundles form a network throughout the endosperm, which enmeshes the endosperm nuclei and supports nuclear migration (Brown et al. 2003; Barranco-Guzmán et al. 2019; Ali et al. 2023). A network of actin bundles was observed in C. mackenii and was connected to the endosperm nuclei (Fig. 4). In some embryo sacs, the actin bundles were not detected in endosperm derived from pollen treated with carbon-ion irradiation (Fig. 4B), and this type of endosperm was also observed in Ar-ion beam irradiation (data not shown). The lack of nuclei diffusing into the endosperm may be related to actin accumulation. Conversely, endosperm with enlarged nuclei formed actin bundles around the nuclei (Fig. 4C, D), suggesting that abnormal division does not necessarily prevent the formation of actin bundles. Some immature seeds derived from fertilization of the irradiated male gametes possibly degenerated at 14 DAP; further analysis of the relationship between endosperm development and the cytoskeleton is needed.

This study revealed that an argon-ion beam is more effective than a carbon-ion beam for the induction of the distinctive embryo and endosperm development. Since various DNA content levels were detected in the embryo and endosperm nuclei derived from pollen treated with carbon-ion irradiation (Hirano et al. 2024), plants regenerated from the embryo and endosperm are expected to be used for basic research and other applications, including breeding. The argon-ion beam-irradiated pollen will have a wide range of applications in the future.

Acknowledgments

This experiment was performed at the RIBF operated by the RIKEN Nishina Center and Center for Nuclear Study (CNS) at the University of Tokyo. This research was supported by JSPS KAKENHI Grant Numbers JP17K15223 and JP20K06035.

Author contributions

TH and HK conceived and designed the research. MS, YK, MM, TH, and TA conducted the experiments. MS, YK, MM, TH, and HK analyzed the data. MS and TH wrote the manuscript. All authors read and approved the manuscript.

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