Studies On The Cytological And Regulation Mechanisms Of Paternal Mitochondrial Inheritance In Cucumber

Mitochondria,which have their own genomic DNA,are essential organelles within eukaryotic cells,which inherited in a non-Mendelian manner.Mitochondrial inheritance is a long-standing scientific problem,which has an important theoretical and practical significance in the fields of genetic diversity,genetic improvement and agricultural production.Despite of the general acception of such inheritance phenomenon,its cellular and molecular regulation mechanisms are still little-known.In angiosperm,the regulation of mtDNA levels during the development of male gamete is the key to mitochondrial inheritance.It is found that the pollen-specific nuclease involved in pollen mtDNA degradation,preventing the paternal mtDNA entering the fertilized eggs in plants with maternal inherited mtDNA,which is believed to contribute maternal inheritance of mitochondria.However,the change laws and the regulation mechanisms of mtDNA levels in the pollen of plants with paternal inherited mtDNA remain unclear.In this study,cucumber(Cucumis sativus L.)was selected as the materials,whose mitochondria are inherited paternally.The mitochondria and its DNA in the pollen development of cucumber were studied by a combination of cytological observation,pollen nuclease expression analysis and transgenic validation.Moreover,the mtDNA in early cucumber embryos was analyzed to detect the fate of maternal mtDNA.By these experiments,the obtained main results are described as follows:1.Cytological studies on the mitochondrial inheritance of cucumberUsing the MTG/DAPI double staining method and electron microscope,the present study provided the evidence that the mitochondria and mtDNA distributed randomly into the vegetative and generative cells in the first microspore mitosis.Though a certain amount of mitochondria with complete structure were present in the vegetative cells,the mtDNA gradually disappeard during the pollen development.Meanwhile,the mitochondria and mtDNA always remained intact in the generative cells,providing the potential for the paternal inheritance of mtDNA;2.Cloning and functional identification of CsDPDl geneBased on homology analysis,cucumber CsDPDl was cloned,which is homolog of the Arabidopsis defective in pollen organelle DNA degradation 1(AtDPD1)gene,playing the direct role in mtDNA degradation.CsDPD1 rescued the Arabidopsis mutant at DPD1,indicating the same function in both plants.Therefore,the degradation of mtDNA in cucumber pollen vegetative cells is likely to be degraded by the exonuclease encoded by CsDPD1,and the altered CsDPD1 expression might account for the retention of mtDNA in the generative cells.3.The regulation of mtDNA levels by CsDPD1 in cucumber pollenUsing qPCR technology,the copy number of mtDNA and the expression of CsDPDl was analyzed during cucumber pollen development.The analysis has revealed that expression of CsDPDl coincides with the decrease of mtDNA in pollen,which was further confirmed CsDPDl plays the direct role in mtDNA degradation in cucumber pollen.While the expression of CsDPD1 in the generative cells is 21 times than the young leaves,it still contains a lot of mtDNA.What's more,we could only detect the paternal origin of mtDNA in the separating cucumber early embryos by PCR,and the maternal mtDNA was degraded in the proembryos.These results indicated that the protection of mtDNA in the generative cell impeding mtDNA degradation by CsDPD1 is a critical factor for paternal mtDNA inheritance.4.The biological function of mtDNA degradation by CsDPDl in cucumber pollen vegetative cellsThe expression levels of CsDPDl were analyzed in different tissues using the qRT-PCR technology,and the relationship between mitochondria and mtDNA were studied in the isolated mitochondria from mature leaves.The results showed that the expression levels of CsDPDl were high in reproductive organs,root and senescence leaves,where nutrient salvage is known to be active,including nucleotide salvage.And mtDNA is not visibly detectable in 3.3%mitochondria from mature leaves,which coincides with the expression levels of CsDPD1 in mature leaves.Taken together with the function of pollen vegetative cells,these analysis enable us to propose that the mtDNA degradation by CsDPD1 in cucumber pollen vegetative cells is likely to contribute to nutrient salvage to support male germ cells in addition to post-pollination development of pollen tubes for fertilization.