Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 51 (2018)

Analysis of DNA damage response in Cyrtanthus pollen after Ar-ion
beam irradiation
T. Hirano,∗1,∗2 A. Hanada,∗1 Y. Hayashi,∗2 T. Abe,∗2 and H. Kunitake∗1
Heavy-ion-beam mutagenesis has been applied forvarious plant materials, and many mutants have successfully been obtained through the screening process.
As one of the plant materials for the irradiation, pollen
was used for mutant induction.1,2) This implies thatthe DNA damage induced by a heavy-ion beam wouldbe repaired during the double-fertilization process, andthe genomic information including mutations would
be transmitted to the next generation. DNA damage response (DDR) in male gametes of Cyrtanthus
mackenii after C-ion beam (22.5 keV/µm) irradiation
has been reported, and the male gametes repaired theDNA lesions during the pollen tube growth.3) In thepresent study, we irradiated mature pollen of C. mackenii with an Ar-ion beam, which has a higher linear
energy transfer (LET) than the C-ion beam, and analyzed the DDR in the male gametes. Moreover, wecompared the DDR after irradiation with Ar-ion andC-ion beams.
Anthers of C. mackenii in 0.2-mL tubes were irradiated with Ar ions (280 keV/ µm) at a dose of 2.5–40 Gy
and then stored at −20◦ C. Pollen grains from the anthers were cultured in 2 mL of liquid pollen culture
medium at 25◦ C in the dark.4) Sperm cell formationin the pollen tube was observed after 4′ ,6-diamidino-2phenylindole staining of the pollen tube. Immunocytochemical analysis for the evaluation of DDR in male gametes was performed according to a protocol describedpreviously.3)
When the germination rate of the pollen grains andpollen tube length were measured after 24 h of in vitro
culture, the germination rate and pollen tube lengthin irradiated pollen showed no decrease compared to
non-irradiated pollen. These results were similar to
those for C-ion irradiation.3) Since C. mackenii forms
bicellular pollen, sperm cells are divided from a generative cell in the pollen tube. Although the sperm cell
formation rate was not decreased up to 10-Gy irradiation, sperm-cell formation was inhibited by high-dose
irradiation of 20 and 40 Gy (Fig. 1). In the C-ionbeam irradiation, the sperm-cell formation rates weredecreased to approximately 70% at 40 Gy and 30% at80 Gy.3) Thus, it is interpreted that the Ar-ion beam
has a greater eﬀect of inhibition of sperm-cell formation compared to the C-ion beam.
To investigate the cause of inhibition of sperm-cellformation, male gametes were isolated from pollen
tubes and immunostained by using anti-α-tubulin antibody for cell-cycle conﬁrmation and by using anti∗1∗2Faculty of Agriculture, University of Miyazaki
RIKEN Nishina Center- 240 -Fig. 1. Sperm cell formation after Ar-ion beam irradiation.
Values ± SD are expressed relative to the unirradiatedcontrol (value set at 100%).

phosphorylated histone H2AX (γH2AX) for the detection of DNA double-strand breaks (DSBs) in thechromosome. After 40-Gy irradiation, the cell cycle
in a part of the generative cells was arrested at themetaphase in pollen mitosis II (PMII) at 24 h of culture. In the male gametes, proportions of metaphasecells were 0% at 0 Gy, 1% at 10 Gy, and 35% at 40 Gy,
suggesting that cell-cycle arrest was induced by thehigh-dose irradiation. The proportions of metaphasecells at 24 h of culture after the C-ion-beam irradiation were approximately 8% at 10 Gy and 11% at40 Gy.3) These results indicated that one of the causesof the inhibition of sperm-cell formation is the cell cycle arrest. Approximately half of the metaphase cellswith 40-Gy Ar-ion irradiation showed γH2AX foci inthe chromosomes, indicating that unrepaired DSBs remained in the genome. Therefore, a spindle assembly
checkpoint in the metaphase would be activated bychromosomal lesions including the DSBs and arrested
in the cell cycle progression.
In the C-ion-beam irradiation, chromosomal bridges
were formed between the sperm cells, and generativecell-like sperm cells, which are deﬁned as cells withgenerative-cell-like nuclei and sperm-cell-like microtubule arrays that completed PMII but failed in chromosome separation, were also formed.3) We also analyzed the male gametes that completed PMII in theAr-ion-beam irradiation, and those data will be usefulfor understanding the eﬀects of heavy-ion-beam irradiation on male gametes.
References
1) K. Naito et al., Genet. 169, 881 (2005).
2) Y. Kazama et al., Sci. Rep. 6, 18917 (2016).
3) T. Hirano et al., AoB Plants 5, plt004 (2011).
4) T. Hirano, Y. Hoshino, J. Plant Res. 122, 225 (2009).