Functional Analysis Of AtPPR2 In Arabidopsis Gametogenesis And Embryogenesis

Angiosperms go through the alteration between the diploid sporophytic phase and haploid gametophytic phase in the life cycle, which is carried out through male /female gametogenesis, double fertilization and embryogenesis. The study of male /female gametogenesis and embryogenesis will be important to understand life cycles and mechanism in angiosperms. Many mutants which phenotypes related with gametogenesis and embryogenesis were found and inditified in Arabidopsis.PPR (Pentatricopeptide repeat protein) mainly reside in either mitochondria and plastid/chloroplast, which are characterized by tandem repeats of a degenerate 35 amino acid motif. PPR family is one of the biggest families in the plants, for example, Arabidopsis genome encode 466 PPR proteins and rice genome encode 480 PPR proteins. Mutation of PPR genes resulted in various phenotypes in the plants, including malfunction of photothesis, cytoplasm male sterile, embryo lethal, abnormal mature seeds, which indicated that PPR genes involved in all kinds of biological process. Further elucidation of PPR molecular mechanism showed that many PPR proteins involve in various RNA post-transcriptional processes, including RNA editing, splicing, stabilization, and translation.AtPPR2 protein is one member of PPR proteins family, and mutations in AtPPR2 gene have previously been found to cause aborted male and female gametophytes, and embryonic lethality. In our study in this thesis, we indentified the Atppr2 mutant caused by loss-of-function mutation of AtPPR2 through TAIL-PCR method. Both Atppr2 male and female gametophyte are partially aborted because of the arrest of the first mitotic division during both male and female gametogenesis. The irregular cell division in Atppr2 embryos development causes delayed embryogenesis, leading to embryonic lethality. emb2750 is another mutant which T-DNA is inserted in AtPPR2. The emb2750 embryos displayed a similar defect to those of the Atppr2 embryos, including the retardation in development. emb2750 mutation causes defect in embryogenesis but does not affect male or female gametophyte development, indicating that emb2750 is a weak mutant allele and that its partial function is sufficient to support normal gametogensis. Approximately 70% of the emb2750 mutant seeds could germinate on agar plates that contained 1/2 MS salts and 3% sucrose, but they produced albino cotyledons which were often deformed and could not undergo further development after germination. Unlike the chloroplasts in the Wt seedlings, there was no typical chloroplast structure or thylakoid membrane system in chloroplasts of the albino emb2750 cotyledons. Instead, these plastids were vacuolated. In additional, the transcript of AtPPR2 in emb2750 cotyledons was about 60% of that in Wt cotyledons, which suggested that the emb2750 allele produces a truncated transcript, which appears partially functional. The defective phenotypes in both Atppr2 and emb2750 were genetically complemented by the binary expression vector containing AtPPR2 full length DNA sequence in the complementation experiment.Q-PCR analysis and GUS reporter gene detected the AtPPR2 transcripts was expressed in all major organs including roots, leaves, stems, seedlings, flowers and siliques, particularly expressed in the parts that undergo cell division. AtPPR2-EYFP fusion protein was transiently expressed respectively in tobacco leaves (Nicotiana benthamiana) and protoplasts derived from Arabidopsis leaves, which results showed that the eYFP signal is localized in chloroplasts. RNA immunoprecipitation (RIP) and Gel mobility shift assays confirmed that AtPPR2 binds to 23S rRNA which is encode by plastid/chloroplast genome.The AtPPR2-modulated translation process might fine-tune the plastid function (including metabolic pathways) toward meeting the need of dividing cells. Plastid/chloroplast genes mainly involved in plastid/chloroplast biogenesis and photosynthesis in the recent report. Although photosynthesis might not be essential for embryogenesis or gametogenesis, other primary metabolites made in plastids, such as amino acids, fatty acids, vitamins, and phytohormones, might be required for these developmental processes. Therefore, the absences of AtPPR2 will likely lead to general dysfunction of plastid functions and further lead to the defects in Atppr2 gametogenesis and embryogenesis. Further elucidation of the molecular event mediated by AtPPR2 could provide novel insights into the mechanism which controls cell division through plastid /chloroplast function.