The Study Of Plastid Occurrence Frequency In The Sperm Cell Of Arabidopsis Thaliana

As a semi-self organelle with DNA, the plastid is transmitted in non-Mendelian manner in the process of generation alternation. In Arabidopsis thaliana, the evidence shows that the plastid is inherited maternally. However, when increasing the progeny sample number, the low frequency of paternal plastid transmission was still observed (3.9×10-5,3 vs 76825). At present, we know little about whether the leaked paternal plastid comes from the sperm cell or vegetative cell of pollen. In this study, through constructing the transgenic lines visualizing the plastid in sperm cell with fluorescent proteins, we investigated whether the sperm cell contains the hereditable plastid, and obtained the following results:1. Through labeling the plastid with green fluorescent protein (GFP), and employing the aniline blue staining, it was found that the plastid could not be observed in the early generative cell (n=56). In contrast, the GFP-labeled mitochondria were always existent. This suggests that the plastid is excluded from the generative cell at the first mitosis of microspore.2. We constructed a double-labele plant which could visualize the plastid in sperm cell by expressing the GFP-Toc34 fusion protein located in the membrane of plastid and RbcS-tdTomato fusion protein located in the matrix of plastid in sperm cells.3. We investigated plastid occurrence frequency in sperm cells of Arabidopsis using the double-labeled transgenic plants. By observing the total 100,850 sperm cells (50,425 pollens), we found 11 sperm cells with punctate fluorescent markers (the frequency is 1.09×10-4). It suggests that the sperm cell has the potential to transmit the paternal plastid.Our results provide the cytological evidence for the maternal plastid inheritance in Arabidopsis. The plastid exclusion from the early generative cell leads to the no plastid in a large number of generative cell, and thus maternal plastid inheritance. Meanwhile, the fact that few sperm cells own the platids also indicates that the leaked paternal plastid may come from sperm cell. The results provide important guidance for the further study of the mechanism of paternal plastid leakage in Arabidopsis.