Studies On Biological Functions Of TIM9and TIM10Complex During Early Embryo And Emdosperm Development In Arabidopsis

In the life cycle of flowering plants, the sporophytic generation takes up most of the time, and during the critical initiation phase of early sporophyte development, embryo and endosperm play dominant roles in influencing plant growth and development. As an ancient organelle in eucaryon, mitochondria must play a necessary role during this process. Mitochondrial proteins TIM9and TIM10(translocase of the inner membrane9and10), are commonly known as a TIM9:10complex in yeast and human, to be important components in the carrier pathway of mitochondria import apparatus, and shuttle between the outer and inner membrane of mitochondria. However, their functions on plant development are unknown. In this study, by using a series of genetic, cell biology and molecular techniques on Arabidopsis wide-type and mutations, such as plant cross, embryo clearing, transgene, in situ hybridization, qRT-PCR, yeast two-hybrid, CO-IP, BiFC, TEM, embryo isolation, immunofluorescence and so on, we report that AtTIM9and AtTIM10are non-redundantly essential for maintaining mitochondrial function during early embryo and syncytial endosperm divisions in Arabidopsis. The main results are as follows:1. We obtained three Arabidopsis mutant lines from the public mutant collections ABRC and NASC, tim9-1, tim9-2, and tim10mutants, and identified that all of them are heterozygote, and cannot found the homozygote in their progenies. The siliques from the three mutant plants all contained about25%aborted white seeds, and the segregation analysis of their progenies showed that the value of resistant to sensitive seeds was about2:1, indicating that AtTIM9and AtTIM10gene mutations lead to homozygous seed lethal. The results of crosses between wild-type and the three heterozygous mutants showed that the mutant gametophytes were not affected, indicating that the defective fertility in mutants is only caused by the aborted homozygous embryos after fertilization. In addition, all of the tim9-1/+, tim9-2/+and tim10/+could be successfully complemented to become fertile. Thus, AtTIM9and AtTIM10both have an essential function in Arabidopsis embryo development.2. We examined embryos of immature ovules from heterozygous mutant siliques and found that all of tim9-1/+, tim9-2/+, and tim10/+mutants display lethality of the homozygous embryos at the dermatogen stage which is arresting early embryo proper with16-32cells. Tracing the development of embryos in the mutants, there was about a quarter showing delayed growth throughout the whole developmental process and stagnating at16/32stage. Meanwhile, according to the tallied numbers of endosperm free-nuclei, endosperm also showed delayed growth in the early stages following fertilization. We crossed tim9-1/+with tim10/+and obtained the tim9-1/+tim10/+line. The seed abortion rate of tim9-1/+tim10/+was43.52%, which is conforming to the expected42.75%according to the Mendel's law. Although the delayed phenotype was more serious, the aborted embryos still stayed at16/32-cell stage. These results indicated that the embryos and syncytial endosperm of aborted ovules in tim9-1/+, tim9-2/+and tim10/+plants grew more slowly than control ovules and finally arrested.3. The expression patterns of AtTIM9and AtTIM10were first analyzed through Genevestigator expression data, and the results showed that both of them present in all tissues with a relatively higher expression level in seeds. From the Pro AtTIM9::GUS and ProAtTIM10::GUS transplants, we found that AtTIM9and AtTIM10genes express ubiquitously throughout the plant tissues, especially in the actively dividing cells during vegetative growth and some reproductive organs. Meanwhile, AtTIM9and AtTIM10expressed throughout the whole process of embryo and endosperm development. By using the quantitative qRT-PCR assay and in situ hybridization, we also observed that the transcripts of AtTIM9and AtTIM10had a ubiquitous expression in plant tissues, and consistently present in embryo and endosperm, indicating that AtTIM9and AtTIM10both have essential effects on early sporophyte.4. In order to identify the homology of AtTIM9and AtTIM10, we performed alignments of the TIM9and TIM10protein sequences in Arabidopsis and other4eukaryotic species, the result showed that both of TIM9and TIM10have high similarities with these homologs, and have a highly conserved twin CX3C motif. We also had predicated the structure of AtTIM9and AtTIM10, and found the high homology of AtTIM9and AtTIM10in3D structures compared with yeast, indicating that AtTIM9and AtTIM10are conserved proteins among eukaryon.5. To investigate the subcellular localization of AtTIM9and AtTIM10, we performed the transformants of AtTIM9-EGFP and AtTIM10-EGFP, and observed that AtTIM9and AtTIM10were both localize in mitochondria of Arabidopsis mesophyll protoplasts. From the results of yeast two-hybridization, Co-IP and BiFC, AtTIM9and AtTIM10were proved to interact with each other in the Arabidopsis mitochondria, indicating that the two proteins exist as a complex.6. By analyzing the isolated tim9-1and tim10embryos, we observed that the abortion of tim9-l and tim10embryos at the16/32-cell stage was caused by loss of cell viability and cessation of division in the embryo proper region, and that this inactivation was due to the collapse of the mitochondrial structure and a decrease in mitochondrial activity. Our characterization of tim9-1and tim10showed that mitochondrial membrane permeability increased, that cytochrome c was released from mitochondria into the cytoplasm, and that TUNEL signals occurred in the16/32-celled embryo proper. These results indicate that a necrosis-like programmed cell death was initiated in the early sporophytic cells of the mutants. Consequently, AtTIM9and AtTIM10are non-redundantly essential for maintaining mitochondrial function of early embryo proper cells and endosperm free-nuclei; these proteins play critically important roles during sporophyte initiation and development in Arabidopsis.7. In order to research the developmental mechanisms of embryo in Arabidopsis, mutants with random T-DNA insertions were selected as research materials which bought from ABRC. The880T-DNA insertion mutant lines in T1generation were planted and observed, and preliminarily obtained66mutant lines with embryonic phenotype. Furthermore, we obtained29sterile mutant lines that could stably genetic to T3generation. These sterile mutant lines were observed by ovules clearing and divided into two categories:13mutant lines with abnormal embryo,11mutant lines with arrested embryo. Another T-DNA flanking sequence of11mutant lines with ovule space phenotype were isolated by TAIL-PCR technique, and the T-DNA flanking sequences of2mutant lines were successfully isolated. Further researching works on them are in progress.