Structural and functional analysis of the key transcription factor ETHYLENE INSENSITIVE3 in Arabidopsis thaliana

• Main Authors: Sian-Ci Li, 李賢啟
• Other Authors: 王隆祺
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/6twkst

碩士 === 國立中興大學 === 生命科學系所 === 106 === Ethylene is a gaseous plant hormone regulating plant growth, development and stress responses including promotion of flower withering, leaf aging and fruit ripening. When ethylene binds to the receptors, the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) kinase is inactivated and fails to phosphorylate ETHYLENE INSENSITIVE2 (EIN2). Subsequently, the cytosolic C terminus of EIN2 (EIN2Cend) is released by proteolysis and translocated to nucleus where EIN2Cend activates EIN3 and EIL1 (EIN3-LIKE1) by suppressing protein degradation of EIN3/EIL1 that is mediated by the 26S proteasome. EIN3/EIL1 are the key transcription factors to regulate the expression of numerous genes responsive to ethylene in Arabidopsis thaliana. It has been shown that dimerization of EIN3 is important for its transcriptional activity. Previous studies uncovered the core DBD (DNA-binding domain) (174-306 a.a.) of EIN3 consisting of two basic domains (BD), BD-III (238-248 a.a) and BD-IV (265-274 a.a.), and a proline-rich region. The BD-III and BD-IV have a proposed role in mediating protein-DNA interaction (PDI) and the proline-rich region is likely involved in protein-protein interaction (PPI). Interestingly, several mutations disrupting EIN3 function including EIN3P216S, EIN3K245N and EIN3T174A are located within the core DBD of EIN3. However, their roles in PPI and PDI have not been fully examined. In addition, three small molecule compounds denoted by 7922, 2133, 2397 that were identified by chemical screens to disrupt EIN3 dimerization in our laboratory. By molecular docking analysis, I revealed a group of amino acids forming non-covalent hydrogen bonds with the compounds within the EIN3 DBD, which suggests the PPI and/PDI of EIN3 may be affected by the small molecules. To examine the functional relationship of the key residues in EIN3 DBD, I generated substitution mutations of P216, K245, and T174 and their spatially adjacent amino acids based on crystallographic studies. Similarly, substitution mutations were also introduced to the amino acid residues predicted to interact with small molecule compounds. Functional characterization was implemented to examine PDI and PPI of EIN3 by yeast one- and two-hybrid systems, respectively, to analyze transcriptional activity of EIN3 by transient gene expression assays in plant cells and to determine EIN3 functionality by complementation experiments using transgenic Arabidopsis plants. I found several amino acid residues crucially involved in EIN3 function, including W214, W215, P216, G218, Q179, E180, L181, K245, Q294, and E295. For example, EIN3K245A allele disrupts the PDI of EIN3 by Y1H assay but still retains partial function when over-expressed in transgenic plants. One of the new findings in this study is that G218 interacts with compound 2133 and is important for both PPI and PDI of EIN3 that EIN3G218 fails to complement Arabidopsis ein3-1/eil1-1 mutant, which highlights the molecular mechanism of small molecules in affecting EIN3 function. In summary, my research uncovers the mechanistic roles of important residues in EIN3 function, which include not only some of the previously known amino acids, but also those involved in interaction with small molecules disrupting PPI and PDI of EIN3. Studies on structure and activity relationship of EIN3 may provide critical information for further development of effective drugs to downregulate EIN3 function, which has practical applications on post-harvest management for fruit ripening, vegetable storage and shelf life of cut flowers. Furthermore, the methodology to screen and characterize small molecule compounds in this study can be applied to functional studies of other biologically important transcriptional factors in crop plants.