Functionalanalysis Of TRNA Specific Nucleoside M5U54 Modification Gene In Arabidopsis Thaliana

As the key element connecting nucleic acid and protein,tRNA not only plays an important role in the process of protein translation,but also participates in the synthesis of a variety of substances and cell metabolism.tRNA is heavily modified,the large number of post-transcriptional nucleoside modifications derive from the four basic nucleosides:Uridine（U）,Cytidine（C）,Guanosine（G）and Adenosine（A）.The presence of modification endows tRNA with structural and functional diversity,and directly influence translation.5-methyluridine（m⁵U,or T）occurs at the 54^thposition within the T-loop of most tRNAs,Trm A in E.coli and Trm2 in S.cerevisiae are the corresponding methyltransferase mediating m⁵U54 formation.Lack of m⁵U54 results in growth defects in bacteria and yeast.Enzymes responsible for m⁵U54 formation has not been reported in higher plants.In this study,we used genetic and biochemical approach to identify genes that might be responsible for m⁵U54 synthesis in Arabidopsis thaliana,to explore the effect of m⁵U nucleoside modification on plant growth and development.In this study,two candidate genes,AtTrm2a and AtTrm2b,were idenfieid as Trm2p homologs in Arabidopsis thaliana by sequence homology.We explore the relationship between gene expression and m⁵U modification using T-DNA mutants and over-expression transgenic plants.We also investigate the effects of AtTrm2a and AtTrm2b on Arabidopsis growth under normal condition and abiotic stresses.The main results are as follows:1.Bioinformatics analysis showed that the Trm2p homologous genes associated with m⁵U modification in Arabidopsis thaliana were At3g21300（AtTrm2a）and At2g28450（AtTrm2b）.The encoded protein had the same tRNA＿U5-Meth＿Tr methyltransferase domain and similar tertiary structure as Trm2p.2.Gene expression were down-regulated in the T-DNA homozygous mutants of AtTrm2a and AtTrm2b,and the modification level of m⁵U nucleoside was decreased,especially in the double mutants,which indicated functional redundancy in the modification pathway of the two genes.3.The leaf length,leaf width and plant height of T-DNA homozygous mutants were significantly reduced compared with that of the wild type Col.0;The plant height of AtTrm2b overexpressed plants was significantly higher than that of the wild type.4.The root length of single and double mutants was significantly shorter under mannitol stress compared with that of wild type.Heat stress inhibited the seed germination rate both in wild-type and mutants.AtTrm2b mutant seedlings were more sensitive to heat stress compared with wild-type.The response of these two genes under heat stress remains to be further studied.5.Primary results from in vitro methylation assay suggest AtTrm2a could methylate endogenous tRNA_GUG^Hisas substrate,whereas AtTrm2b can methylate tRNA_CAA^Leufor m⁵U54 formation.