Changes In Mitochondrial DNA Levels During Early Embryogenesis In Torenia Fournieri And Arabidopsis Thaliana

Mitochondrial DNA(mtDNA)is the genetic substance carried by mitochondria,which regulates the mitochondrial activity with the nuclear DNA.During mammalian egg cell development,mtDNA increases to>10~5 copies,which is about 100 times that in somatic cells,given that a normal somatic cell contains about 10~3 copies of mtDNA.This abundant mtDNA in egg cells ensures that early embryos can normally develop to the blastocyst even without mtDNA replication.In angiosperms,the amount of mtDNA in egg cells is similar to that in somatic cells,which suggests that,during early embryogenesis in angiosperms,the dynamics of mtDNA may differ from that in mammalian embryo cells.However,due to the difficulty in isolating early embryo cells,the change in the amount of mtDNA has never been investigated.Using Torenia fournieri and Arabidopsis thaliana,we investigated the changes in mtDNA levels during early embryogenesis.Real-time PCR results showed that the mature egg cell and the late zygote of T.fournieri contained 78.0±22.7 and 154.2±36.9 copies of mtDNA,respectively,indicating that mtDNA doubled during zygotic development.We also quantified the number of mtDNA in early basal and apical cells,which contained 103.0±26.1 and 33.4±10.5 copies of mtDNA,respectively.Given that the early apical and basal cells are the division products of the mature zygote,this result actually indicated that the mature zygote had 136.4 copies of mtDNA,which further confirmed that the mtDNA doubled during zygotic development.To provide the cytological evidence of mtDNA doubling,we generated transgenic line ProDD45:MT-GFP,in which the mitochondria in egg cell and zygote were labelled with green fluorescence protein.After staining with 4',6-diamidino-2-phenylindole(DAPI),this line enabled us to discriminate mitochondrial nucleoids from plastid nucleoids.Quantitative fluorescence microscopy showed that,during zygotic development in T.fournieri,the mitochondrial number,mitochondrial nucleoid number,and mitochondrial nucleoid fluorescence increased from 317.3±28.3,220.0±29.2,and 3.2±0.5×10~4 to 584.3±26.5,412.7±13.5,and 7.2±1.5×10~4,respectively,further confirming that mtDNA doubled during zygotic development.Using fluorescence microscopy,we also surveyed the changes in the amount of mtDNA during zygotic development in transgenic line ProDD45:MT-GFP of A.thaliana.Unlike T.fournieri zygote,the mtDNA in A.thaliana zygote did not double in amount,as shown by that the mature egg cell and mature zygote contained 355.4±31.8 and 326.2±28.1 mitochondria,253.8±20.0 and 254.0±33.8 mitochondrial nucleoids,and 3.1±0.3×10~4 and 3.7±0.5×10~4mitochondrial nucleoid fluorescence,respectively.However,during two cell embryo development,we found that mtDNA doubled.The mitochondrial number,mitochondrial nucleoid number,and mitochondrial nucleoid fluorescence increased from 132.8±21.4,99.3±18.6,and 1.4±0.2×10~4 to 211.7±20.8,183.3±11.5,and 2.6±0.2×10~4 during apical cell development.These findings indicate that mtDNA doubles during early embryogenesis in T.fournieri and A.thaliana,revealing that mtDNA in early embryos of angiosperms has different dynamics from that in early mammalian embryos.We also investigated the distribution of mtDNA in the mature zygotes of T.fournieri and A.thaliana.Using the hypothetical division plate at the midline of the nucleus of the mature zygote,we divided them into two parts:the apical and basal parts.The number of mitochondrial nucleoid and the mitochondrial nucleoid fluorescence were calculated.In T.fournieri,the ratio of the mitochondrial nucleoid number in the basal part to that in the apical part was 2.96:1,and the ratio of the mitochondrial nucleoid fluorescence in the basal part to that in the apical part was 3.00:1.In A.thaliana,the ratios were 1.68:1 and 1.64:1,respectively.Therefore,the distribution of mtDNA in the mature zygote of T.fournieri differs from that in the mature zygote of A.thaliana.Since the basal and apical parts of the zygote will develop into the basal and apical cells,these findings suggest that mtDNA will be allocated into basal and apical cells with the ratios of 3.00:1 in T.fournieri and 1.64:1 in A.thaliana.Interestingly,this mtDNA distribution makes the apical cells of T.fournieri and A.thaliana have similar mtDNA amount and the basal cells have largely different mtDNA amount.The biological significance of this phenomenon is currently unclear and needs further investigations.In summary,these findings first reveal the changes in mtDNA levels during early embryogenesis in angiosperms.Moreover,we also show that the mtDNA distribution in the mature zygotes is different in different species,providing evidences for understanding early embryogenesis in angiosperms.