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DNA damage response in male gametes of Cyrtanthus mackenii during pollen tube growth

机译：花粉管生长过程中美贯叶拟南芥雄配子的DNA损伤反应

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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.
机译：植物的雄配子体在双重受精过程中会受到环境压力和诱变剂的影响，因此需要修复DNA损伤才能将基因组信息传递给下一代。然而，雄配子的DNA损伤反应仍不清楚。在本研究中，我们分析了在花粉管生长过程中，美兰特兰贯叶生殖细胞对DNA损伤的反应。用能诱导DNA双链断裂（DSB）的碳离子束辐照双细胞花粉，然后在液体培养基中培养辐照的花粉粒。从培养的花粉管中分离出雄配子，并用于免疫荧光分析。尽管辐照后未观察到对花粉管生长的抑制作用，但大剂量辐照后精子细胞的形成明显减少。大剂量照射后，生殖细胞的细胞周期进程被阻止在花粉有丝分裂II的中期，并且在大多数被捕的细胞中检测到磷酸化的H2AX（γH2AX）病灶（DSB的指示剂）。但是，这些病灶在中期之后的细胞中未检测到。受辐照的生殖细胞中的细胞周期进程受纺锤体装配检查点调节，并确认了围绕DSB的组蛋白的修饰。这些结果表明，在花粉管生长过程中，生殖细胞可以利用DNA损伤反应途径识别和处理基因组损伤。此外，随着培养时间的延长，具有γH2AX病灶的生殖细胞数量减少，这表明生殖细胞中的DSB被修复。

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