The Preliminary Function Studies On Chloroplast Proteins And N-Terminal Acetyltransferases Genes In Arabidopsis

Embryogenesis is the beginning of plant individual development,the cell differentiation and pattern formation during embryo development lays the foundation for development of mature seeds and production of complete plants.Arabidopsis thaliana is a major model plant for genetic research.Recently,a large number of studies have revealed the genetic pathways and molecular mechanisms in embryo development of Arabidopsis.However,little is known about the regulatory machnism and networks as well as signal communications between different tissues and cells during embryo development of higher plants.Chloroplast is an essential semi-autonomous organelle in plants.It is not only the place for photosynthesis,but also responsible for synthesizing and storaging important compounds.The normal development of chloroplasts is closely related to the morphogenesis of plant embryos.However,it is still not clear how the chloroplast genes and proteins function in embryo development.N-terminal acetylation catalysed by N-terminal acetyltransferases is a highly abundant protein modification in eukaryotes,which play important roles in protein degradation,localization,complex formation and interaction between protein and membrane system.The previous researches of N-terminal acetylation are mainly concentrated on the species of human and yeast,however,poorly is known about its function in plant growth and development.In this study,we tried to reveal the functions of three genes encoding chloroplast proteins in embryo development of Arabidopsis by using molecular biology,genetics,cell biology,bioinformatics and many other techniques.In addition,we analysed the expression and evolution of eight genes encoding N-terminal acetyltransferases in Arabidopsis.The main results obtained are as follows:1.We obtained five T-DNA insertion mutants(em30,em33-1,em33-2,em54-1 and em54-2)of three EM(EMBYRO-DEFECTIVE)genes in Arabidopsis,and found that they were all heterozygotes by PCR assay.About 25%white ovules were found in the siliques of five heterozygotes,and finally aborted.Genetic assay showed that the ratios of with T-DNA insertion and without insertion were close to 2:1 in the progenies of five mutants.Nevertheless,the transmission efficiencies of the female and male gametophytes in five mutants were normal,indicating that T-DNA insertions in five mutants couldn't affect the transmission of gametophytes but lead to homozygous seed lethal.Ovule clearing and embryo observation of em30,em33-1 and em33-2 displayed two kinds of aberrant embryos:one kind was irregular globular embryos with disordered split surface,while another kind was abnormal cotyledon embryos which had an enlarged angle between two cotyledons.The embryo development of em54-1 and em54-2 were both arrested at globular stage and the split surface of embryos was also aberrant.The complemental assay by transgenic technique showed that EM30,EM33 and EM54 could rescue the aborted embryos completely,confirming that the mutations of EM genes were exactly the causes of embryonic lethality.Through quantitative PCR and promoter fused with GUS,we found that EM genes expressed widely in different tissues and reproductive organs.In order to reveal whether EM30,EM33 and EM54 are chloroplast proteins,the protoplast system was used to perform subcellular localization experiments and these three EM proteins all were found to be located in chloroplast.Homologous comparison and phylogenetic analysis turned out that EM30 was a homologous protein of DEA(D/H)-box RNA helicase family,EM33 and EM54 belonged to the PORR and YGGT protein families,respectively.Three EM proteins all displayed a deep degree of homology between different species in system evolution.The expression of EM genes under different light conditions were detected by quantitative PCR,showing that light signal is an important regulatory factor of theEM genes expression.2.Twelve T-DNA insertion mutants of eight NAA(N-ALPHA ACETYLTRANSFERASE)genes were obtained from the ABRC,and genotypic analysis showed that homozygous plants could be obtained except naa50-1/+ and naa50-2/+.We observed the development processes of these mutants and found that the seedlings of naa30-1 and naa30-2 grew slower than wild-type,while other mutants had no vegetative growth defects.In addition,all twelve mutants had no reproductive defects.The transcriptional levels of NAA genes in homozygous mutants were detected,the results turned out that the expression levels of corresponding genes in naa20,naa25-1,naa30-1 and naa40-1 were not changed,while in naa25-2,naa30-2,naa38,naa40-2,naa60-1 and naa60-2 were down-regulated,indicating that T-DNA insertions of 10 homozygous mutants didn't result in completely silence of corresponding genes.Through RT-PCR technique,we found that NAA genes are expressed widely in different tissues and reproductive organs.Interestingly,eight NAA genes consistently displayed a high level of expression in seedlings,inflorescences and flowers.Phylogenetic trees were constructed to analyze the homology of NAA proteins in eukaryotes.Evolutionary analysis showed that NAA proteins were highly conserved in plants,animals and yeasts.In addition,the phylogenetic trees of the eight NAA proteins also had some similarities,indicating that the NAA genes were also conservative in evolutionary process.