Genetic Screening for Cellular Redox Regulators and Their Functional Analysis in Plant

다국어 초록 (Multilingual Abstract)

Recently, it has been reported that Bax, a mammalian pro-apoptotic member of the Bcl-2 family, can induces cell death when expressed in yeast. To investigate whether Bax expression can induce cell death in plant, we produced transgenic Arabidopsis plants that contained murine Bax cDNA under control of a glucocorticoid-inducible promoter. Transgenic plants treated with dexamethasone(DEX), a strong synthetic glucocorticoid, induced Bax accumulation and cell death. These observations suggest that some elements of cell death mechanism by Bax may be conserved among various organisms. Therefore, we employed yeast, a powerful genetic tool, to identify the molecular determinants of Bax-induced apoptosis in plants. Soybean and Arabidopsis cDNA library was co-transformed with the Bax gene into yeast cells, and over-expressed genes that could suppress Bax-induced cell death were isolated. From the repertoire of the Bax-inhibiting proteins obtained, we characterized ascorbate peroxidase(sAPX) and nucleoside diphosphate kinase(NDPK) in detail. Examination of reactive oxygen species(ROS) production using the fluorescence method of dihydrorhodamine123 oxidation revealed that expression of Bax in yeast cells generated ROS, which was greatly reduced by co-expression with sAPX, suggesting that sAPX inhibits the generation of ROS by Bax, which in turn suppresses Bax-induced cell death in yeast. These results indicate that this system maybe useful yeast-based genetic screening method to clone plant genes involved in cellular redox regulation. Therefore, we examined the functional significance of nucleoside diphosphate kinase(NDPK) as redox regulator in plant.
H₂O₂ stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana(AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDP kinase activity, and they have lower levels of reactive oxygen species(ROS) than wildtype plants. Mutants lacking AtNDPK2 had higher levels of ROS than wildtype. H₂O₂ treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H₂O₂-activated A. thaliana mitogen-activated protein kinases. In the absence of H₂O₂ treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein(MBP) phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a novel regulatory role in H₂O₂-mediated MAPK signaling in plants.