Study On The Cell Phase Of Mature Gametes Of Arabidopsis Thaliana And Torenia Fournieri

Cell cycle contains four phases: G1,S,G2 and M,among which the typical cytological event in S phase is the replication of nuclear DNA.During the proliferation of most cells,the four phases are continuous.However,it is not the fact for the mature sperm and egg cells.In mammals,mature sperm cells arrest the cell cycle at G1 phase,while mature egg cells do at meiosis II.Fertilization activates the egg cells and causes them to complete the meiosis to form a zygote,in which the nuclear DNA from the sperm and egg cells initiates the replication.However,in flowering plants,it is debatable which phase the mature sperm and egg cells arrest at? For example,DAPI staining shows that the sperm and egg cells of barley and rice arrest the cell cycle at G1 phase before fertilization,and the sperm and egg cells of A.thaliana and tobacco do at G2 phase before fertilization.These different experimental results prompted us to reassess the cell cycle phase of the mature sperm and egg cells of tobacco,A.thaliana and T.fournieri using EdU staining in this study,and the following results were obtained:(1)DAPI staining combined with laser confocal microscopy were used to measure the changes in the nuclear DNA content of T.fournieri sperm cells and zygotes during their development.The statistics results showed that the total fluorescence value increased by about 7 times during sperm cell development.During zygote development,compared with that in mature egg cells,the total fluorescence value in mature zygotes increased about70%.Given that only a one-fold increase should be observed even if the sperm cells replicated their nuclear DNA,these results suggested that DAPI staining is not suitable for measuring the changes in the DNA content during gametogenesis.(2)Using early tobacco pollen grains as experimental materials,this study confirmed that EdU staining could detect the nuclear DNA replication in generative cells.In three separate experiments,3-5% generative cells exhibited an EdU staining signal.The signal occured only in generative cells but never in pollen vegetative cells.(3)EdU staining was used to investigate whether the nuclear DNA replication occurred during sperm cell development in tobacco,A.thaliana and T.fournieri.In tobacco,none ofthe sperm cells exhibited an EdU staining signal even the sperm cells at seven different developmental stages were examined(n=531?504?507?524?504?569?572).Similarly,the EdU staining signal was either not present in A.thaliana sperm cells at four developmental stages(n=699?592?573?508)and in T.fournieri sperm cells at three developmental stages(n=703?551?522).These results suggested that sperm cells of all three species did not replicate the nuclear DNA during development.(4)This study also investigated whether the nuclear DNA replication occurred during egg cell development in A.thaliana and T.fournieri by the EdU staining method.The results showed that none of the early(n=1876)and mature(n=2213)A.thaliana egg cells showed the EdU staining signal.Similarly,the EdU staining signal was either not present in the early(n= 2613)and late(n= 4301)egg cells of T.fournieri.These results indicated that the egg cells either did not replicate the nuclear DNA during development.(5)The results that the sperm and egg cells did not replicate their nuclear DNA indicated that the zygotes had to replicate the nuclear DNA before division.In fact,we did observe the nuclear DNA replication during zygote development in A.thaliana and T.fournieri using EdU staining.Specifically,among 705 A.thaliana zygotes,7 zygotes exhibited the obvious EdU staining signal and among 3119 T.fournieri zygotes,12 zygotes showed the obvious EdU staining signal.(6)In order to provide other evidence to support the nuclear DNA replication in zygotes,the transgenic marker Pro HTR2:CDT1A(C3)-td Tomato of A.thaliana was generated in this study.The results showed that this marker could effectively indicate the S and G2 phases in the root tip cells.Real-time observation showed that the accumulation of fluorescent proteins in the nucleus occured after the cells entered the S phase.Therefore,combined with real-time observation,this marker can be used to indicate the S phase of the cell cycle.Tanken together,the results of this study show that nuclear DNA replication do not occur during gamete development in A.thaliana and T.fournieri.In contrast,the nuclear DNA replication occurs only during zygote development.These results suggest that the G1-phase-gamete-participated-in fertilization is present at least in A.thaliana and T.fournieri.