Abstract
Male gametophytes of plants are exposed to environmental stress and mutagenic agents during the double fertilization process and therefore need to repair the DNA damage in order to transmit the genomic information to the next generation. However, the DNA damage response in male gametes is still unclear. In the present study, we analysed the response to DNA damage in the generative cells of Cyrtanthus mackenii during pollen tube growth. A carbon ion beam, which can induce DNA double-strand breaks (DSBs), was used to irradiate the bicellular pollen, and then the irradiated pollen grains were cultured in a liquid culture medium. The male gametes were isolated from the cultured pollen tubes and used for immunofluorescence analysis. Although inhibitory effects on pollen tube growth were not observed after irradiation, sperm cell formation decreased significantly after high-dose irradiation. After high-dose irradiation, the cell cycle progression of generative cells was arrested at metaphase in pollen mitosis II, and phosphorylated H2AX (γH2AX) foci, an indicator of DSBs, were detected in the majority of the arrested cells. However, these foci were not detected in cells that were past metaphase. Cell cycle progression in irradiated generative cells is regulated by the spindle assembly checkpoint, and modification of the histones surrounding the DSBs was confirmed. These results indicate that during pollen tube growth generative cells can recognize and manage genomic lesions using DNA damage response pathways. In addition, the number of generative cells with γH2AX foci decreased with culture prolongation, suggesting that the DSBs in the generative cells are repaired.
Highlights
In flowering plants, male gametophytes transport pairs of sperm cells by elongating pollen tubes into female gametophytes, which contain egg cells and central cells
Transcriptome analysis in sperm cells has revealed that DNA repair or DNA damage response (DDR) genes are expressed in sperm cells, and the repair-related genes are enriched in the sperm cells compared with pollen (Engel et al 2003; Borges et al 2008)
The germination rate of non-irradiated pollen grains reached a plateau at up to 3 h of culture, and the germination rate measured at 24 h was not higher than that measured at 3 h (Fig. 1A)
Summary
Male gametophytes (pollen grains) transport pairs of sperm cells by elongating pollen tubes into female gametophytes (embryo sacs), which contain egg cells and central cells (reviewed in Lord and Russell 2002). When mature pollen from Petunia hybrida was UV-irradiated, unscheduled labelling of pollen DNA by 3H-thymidine was observed during pollen germination (Jackson and Linskens 1978) This observation suggests that a repair-like DNA synthesis is induced in the pollen DNA in response to UV irradiation. Transcriptome analysis in sperm cells has revealed that DNA repair or DNA damage response (DDR) genes are expressed in sperm cells, and the repair-related genes are enriched in the sperm cells compared with pollen (Engel et al 2003; Borges et al 2008). These findings indicate that DNA repair pathways function in male germline cells
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