A Preliminary Investigation Of AtSIRT1 In Arabidopsis Thaliana

Sirtuins have emerged as important proteins in aging, stress resistance and metabolic regulation. Mammals contain seven sirtuins (SIRT1-7), but Arabidopsis thaliana contain 2 homologous proteins, named AtSIRT1 (At5g09230) and AtSIRT2 (At5g55760). In this study, we pay attention to AtSIRTl, a locus encoding mitochondrial protein.AtSIRT1 has 7 transcripts, and processes 4 mature mRNA, named S1.3, S1.4, S1.5 and S1.7. Semi-quantitative RT-PCR analysis revealed that, the expression levels of these transcripts in different tissues differ. Promoter analysis showed that AtSIRT1 strongly expressed in meristem tissues, and the expression was under light regulation. Using Gateway recombinant cloning system, Arabidopsis plant expressing N-termined GFP fusion proteins were generated. We found that S1.4-GFP was located to the nucleus, and the others were located to the mitochondria. Four transcripts were overexpressed harbouring the FLAG tag. We choose S1.3-FLAG and S1.7-FLAG for further study here. In addition, we characterized two T-DNA insertional mutants of AtSIRTl.Transgenic plants overexpressing the S1.7-FLAG displayed, low seed germination rate and delayed seedling development. When they grow on B5 medium supplemented with high levels of sucrose (e.g 150 mM), they fail to develop green expanded cotyledons and true leaves, and even die. Transgenic plants overexpressing S1.3-FLAG and the T-DNA mutants exhibited vigorous growth both on B5 and high levels of sucrose media. The glucose contents in these seedlings were determinated. The T-DNA insertional mutants had slightly lower glucose contents in comparison with wild-type. While the S1.7-FLAG line had significantly lower contents of glucose in comparison with wild-type.Determination of the isoenzyme activity of glutamate dehydrogenase (GDH) revealed that the S1.7-FLAG seedlings had significantly higher GDH activity than wild-type when they grow on B5 and high sucrose media. The GDH activity in the T-DNA mutants was slightly lower than wild-type when they grow on B5 medium, and displayed no difference with wild-type when they grow on the high sucrose medium. The activity of GDH in S1.3-FLAG seedlings was much lower than wild-type.We suggested that the phenotype of S1.7-FLAG may resulted from increased GDH activity and alternated carbon skeleton supply. The phenotype of S1.3-FLAG may resulted from defect of the function of S1.7.