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Subcellular Localization and Functional Analysis of SIRT Gene Products in Arabidopsis in Arabidopsis Thaliana

Author: ChenYong
Tutor: ZhuMuYuan
School: Zhejiang University
Course: Genetics
Keywords: Arabidopsis AtSIRT1 AtSIRT2 phylogenetic tree glutamatedehydrogenase mitochondria aging bimolecular fluorescence complementation DNA double strand breaks hSIRT4 hSIRT6
CLC: Q943.2
Type: PhD thesis
Year: 2012
Downloads: 106
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Abstract

Sirtuins (Sir2) are NAD-dependent deacetylases and/or ADP-ribosyltransferases that are highly conserved from bacteria to human. Sir2was originally shown to extend lifespan in budding yeast. In mammals, there are seven sirtuins (SIRT1-7). All mammalian sirtuins contain a conserved NAD-binding and catalytic domain, termed as the sirtuin core domain, but differ in their N and C-terminal domains. They have different specific substrates and biological functions, and are found in various cell compartments. Sirtuins have been increasingly recognized as crucial regulators for a variety of cellular processes, ranging from energy metabolism and stress response to tumorigenesis and aging.hSIRT4is found in the mitochondrial matrix. The only reported activity of hSIRT4is the NAD+-dependant ADP-ribosyltransferase activity. It has been shown that hSIRT4can ADP-ribosylate glutamate dehydrogenase (GDH) and control insulin secretion in pancreatic β-cells in response to CR. hSIRT6is a nuclear, chromatin-bound protein. hSIRT6is shown to have ADP-ribosyltransferase and deacetylase activity, preferentially acting on histone H3K9and H3K56. hSIRT6is playing an important role in DNA repair, telomere function, genomic stability, and cellular senescence. SIRT6is involved in DNA double-strand break repair by regulating C-terminal binding protein (CtBP), DNA protein kinase and poly[adenosine diphosphate (ADP)-ribose] polymerase1(PARP1).Arabidopsis thaliana contain two SIRT gene families, named AtSIRTl (At5g09230) and AtSIRT2(At5g55760). However, little research on SIRT in plant has been conducted. The present study focuses on the AtSIRTl and AtSIRT2of Arabidopsis. Here, we aim to reveal their expression patterns, subcellular locations and function in plants. The results are summarized as follows:1Phylogenetic trees of AtSIRTl, AtSIRT2and the homologous proteins of other model organisms is constructed by means of the MEGA method. The results show that AtSIRTl is closest homologs with mammalian SIRT4, and AtSIRT2is closest homologs with mammalian SIRT6. 2The AtSIRT-EGFP fusions are constructed to analyze the subcellular localization of AtSIRT proteins. The results suggest that AtSIRTl was targeted to the mitochondrial, and AtSIRT2was located in nucleolus. Meanwhile, PAtSIRT1:GUS report lines were generated. The histochemical localization of GUS staining indicated that AtSIRT1is expressed in roots, stems and leaves, especially in the leaf and root tips. Investigation of accumulation of AtSIRTl transcripts during a photoperiod by real-time PCR indicated that AtSIRT1gene was a diurnal-regulated gene.3We characterized the Arabidopsis T-DNA insertional mutant lines of AtSIRT. We found that mutation in AtSIRT1leaded to early senescence of cotyledons. Four transcripts of AtSIRTl were overexpressed harbouring the FLAG tag. Transgenic plants overexpressing the AtSIRT1.7-FLAG displayed low seed germination rate and delayed seedling development. When they grow on B5medium supplemented with high levels of sucrose, they fail to develop green expanded cotyledons and true leaves, and even die. Transgenic plants overexpressing AtSIRT1.3-FLAG and the T-DNA mutants exhibited vigorous growth both on B5and high levels of sucrose media. Sirtinol, a inhibitor of SIRT which did not cause the same phenotype of the mutation of AtSIRTl indicated that the mechanism of Sirtinol in Arabidopsis was different from other organism.4Bimolecular fluorescence complementation (BiFC) means two non-fluorescent complementary fragments of fluorescent protein can reassemble to form fluorescent complex and restore fluorescence when they are fused to two proteins that interact with each other. To figure out which protein AtSIRT2does to interact with, we construct a series vectors mat associate with mammalian hSIRT6, including DNA-PK-cGFP, CtIP-cGFP and PARP1-cGFP. Protein-protein interaction between AtSIRT2and substrates were studied in vivo. The results suggest that PARP1was the substrate.5The DNA double strand breaks (DSBs) is one o f the most serious form of DNA damage. Nonhomologous end joining (NHEJ) and homologous recombnation (HR) are the two major pathway to repair DNA double strand break damages in both higher plants and animals. To check the role of AtSIRT2during DSB repair of Arabidopsis, we constructed a series reporter vectors, including EJ2-GFP, EJ5-GFP, HDR-GFP. The results show that AtSIRT2is playing an important role in DNA repair.AtSIRTl was located in mitochondria as hSIRT4of human, and maybe take part in respiration and electron transformation chain to regulate cotyledon and leaf senescence. AtSIRT2was located in nucleolus as hSIRT6of human, maybe play important role in DSB repair.

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