Studies On Biological Function Of Translocase Of The Outer Membrane 40 In The Embryonic Development Of Arabidopsis Thaliana

Double fertilization is a landmark event in the development of angiosperms.In the process in double fertilization in dicotyledonous plants,one sperm cell and one egg cell fuse to form a zygote,and to develop into embryo after a series of cell division and differentiation.The other sperm fuses with the central cell to form the endosperm,which undergoes the syncytium and cellularization and nourish the embryo growth,until it degrades and disappears at the late stage of development.Early embryogenesis formed the precursor of the basic organization,in the past decades,scientists made deep research in the mechanism of embryonic development in Arabidopsis,and found that the process of embryogenesis and pattern formation is highly programmed.The events are regulated by a precise and complex gene expression network,and a series of genes were involved in the process of embryonic development and pattern formation in Arabidopsis.Mitochondrial Translocase of the Outer Membrane(TOM)transports the protein precursor synthesized in the cytoplasm into the mitochondria and acts as an entry gate.Previous studies in the biological function of TOM40 protein in different species have shown that the expression level of the protein in yeast is related to the release of oxygen ions,and TOM40 has also been proved to be related to aging and disease in human cells.However,little is known about the biological function of TOM40 in model plant Arabidopsis.Therefore,two mutants of Arabidopsis tom40 were used as materials in this study,and by using bioinformatic,molecular biology,cellular biology,genetics and other techniques,it was found that the mutation of AtTOM40 gene caused mitochondrial structure disorder,resulting in abnormal embryonic development,and finally caused seed abortion.At the same time,the interaction between the subunits of TOM complex in Arabidopsis was studied by yeast double hybridization,which provided experimental basis and new information to clarify the mechanism of how TOM40 works in Arabidopsis and had important theoretical significance.The main results obtained in this study are as follows.1.Firstly,bioinformatics software CLUSTALX(Ver 1.83),MEGA4,Swiss-PdbViewer 4.01,PyMOL 1.7,and online database(http://www.arabidopsis.org,http://blast.ncbi.nlm.nih.gov,http://swissmodel.expasy.org,http://swissmodel.expasy.org,https://www.ebi.ac.uk/interpro/sequencesearch,http://ibs.biocuckoo.org were used to analyze the TOM40 protein in different species.The results showed that the amino acid structure and sequence of TOM40 protein in different eukaryotes were conservative,and the AtTOM40 protein contained 19 ?-stranded fragments domains and 2 ?-helix structures,which had the highest homology with that in Brassica.Using yeast TOM40 as template,homologous modeling was performed for the three dimensional structure of TOM40 in dicotyledon Arabidopsis and monocotyledon Oryza.The main structure of TOM40 proteins in the three species basically overlapped,all of them contained 19 stranded ?-barrel proteins,and the protein structure of Arabidopsis and Oryza contained 2 ?-helix structures.Further structure prediction about TOM40 proteins in different species showed that TOM40 is a kind of pore-forming proteins in eukaryotes,with a hole in TOM40 structure in different species,and the size ranged from 308 amino acids in Arabidopsis to 387 amino acids in yeast,implying that the function of this conserved porin domain is to help the unfolded polypeptide chain transport into mitochondria.The results above indicated that TOM40 is a conserved protein that performs basic biological functions in organisms.2.Two T-DNA insertion mutants SALK₁28170(tom40-1/+)and CS873594(tom40-2/+)were obtained from ABRC(Arabidopsis biological resource center,http://abrc.osu.edu/),and were verified and analyzed.Three primers were used to identify the T-DNA flanking sequences of the two mutants,and the results showed that the T-DNA of tom40-1 mutant was located on the second exon of AtTOM40 gene,and the T-DNA of tom40-2 mutant was located on the seventh exon of AtTOM40 gene.Subsequently,the two mutants were screened and identified,and it was found that they were both heterozygous mutants and no homozygous descendants could be obtained.At the same time,the development of both male and female gametophytes was not affected in the two mutants,but abnormal albino ovules were observed in mature siliques and eventually died.The seed abortion rate in the two mutants was 24.92% and 25.04%,respectively,indicating that the mutation of AtTOM40 gene resulted in the death of embryo recessive homozygous.The complementation analysis confirmed that the lethal embryonic phenotype of both mutants could be completely rescued,indicating that the mutation of AtTOM40 gene was the cause of seed abortion.3.Ovules in the two mutants were observed by clearing technique,the results showed that the development of the embryos was abnormal from the heart stage,and they could not produce normal cotyledon,apical meristem and other organ primordia,which was similar to the embryo status at the globular stage.The height and width of the embryo proper in the two mutants were measured,and the results showed that the size of the embryo proper would increase with the development process,but would eventually arrest.According to the statistics of the embryonic development process of the wild type and the two mutants,the results showed that the proportion of the two mutants similar to the globular embryo was higher and higher with their development process,and the final proportion was 24.39% and 24.49% respectively,which was equivalent to the abortion rate.The endosperm of tom40-1 mutant showed normal free nuclear division and cellularization.The above results confirmed that the mutation of AtTOM40 gene would lead to abnormal embryonic development and seed abortion,therefore,the gene plays important role in the embryonic development of Arabidopsis.4.Quantitative real-time PCR technique was used to detect the transcriptional expression level of AtTOM40 gene,and the results showed that the gene was expressed in root,stem,leaf,flower,inflorescence,siliques at 1/3/5/7 days after pollination(DAP)and seedlings at 7 and 14 days after germination,among which the expression level was relatively high in mature flowers,inflorescence and siliques at 1 day after pollination.GUS signal was detected by fusing the AtTOM40 promoter with GUS protein,and the results showed that the GUS signal could be detected in mature leaves,trichomes,7-day germination seedlings and inflorescences,especially in the cotyledons of mature embryos.However,GUS signal in reproductive organs such as anthers,filaments and stigma was weak or even undetectable.It was speculated that AtTOM40 gene was involved in the process of nutritional growth,embryonic development and organ primordial differentiation in Arabidopsis,but not in the developmental process of male and female gametophytes.In situ hybridization was used to further study the expression pattern of AtTOM40 gene in Arabidopsis embryos,the results showed that AtTOM40 gene was involved in the developmental process of Arabidopsis embryos and endosperm.5.The AtTOM40 promoter was fused with GFP protein and the mesophyll protoplast was stained by Mitotracker Red.GFP fluorescence localization and Mitotracker Red staining in protoplasts of transgenic plants were observed by using confocal laser scanning microscopy(CLSM).The results showed that there was co-localization of AtTOM40 protein and mitochondrial fluorescence in protoplasts of transgenic plants,indicating that the protein localizes in mitochondria.The ultrastructure of mitochondria was observed by ultrathin section and transmission electron microscopy(TEM),and it was found that no regular mitochondrial cristae structure could be observed in embryo proper cells of tom40-1 mutant.These results demonstrated that the AtTOM40 gene located in mitochondria in Arabidopsis,and the gene encoded a mitochondrial protein,which was consistent with the bioinformatics prediction results.In the mean time,mutation of the AtTOM40 gene will affect the mitochondrial structure in embryo proper cells,indicating this gene is involved in the biogenesis of mitochondria,thus regulates the embryonic development in Arabidopsis.6.In order to further study the biological function of AtTOM40 gene,the expression of 19 Arabidopsis outer mitochondrial membrane(OMM)protein-related genes was detected by using quantitative RT-PCR.The 6 DAP ovules in wild type and 6 DAP albino ovules in tom40-1/+ plants were collected,respectively.The results showed that there were significant changes in the expression levels of 14 genes,which proved that the mutation of AtTOM40 gene affected the function of outer mitochondrial membrane proteins at the transcriptional level,thus disturbing the biogenesis of mitochondria.Expression levels of 19 genes related to embryonic development and pattern formation in Arabidopsis were also detected,and significant changes were detected at the expression levels of 18 genes,indicating that the mutation of AtTOM40 gene resulted in changes at the transcription level of embryonic development and pattern formation7.The interaction between the TOM40 subunit and other subunits of TOM complex in Arabidopsis was examined by using Yeast two-hybrid technique,the results showed that TOM40 could interact with TOM5 and TOM9,and TOM5 could interact with TOM7-1,TOM7-2,TOM9 and TOM40.There is interaction between TOM6 and TOM7-1.As well as between tom7-1 and tom7-2.And TOM9 interacts with tom7-1 and TOM40.Based on the experimental results,a schematic diagram of the interaction between the subunits of Arabidopsis TOM complex was drawn,which could provide information for revealing the mechanism of TOM complex in the transport process of mitochondrial outer membrane protein.