Roles of AtMAPR2 in brassinosteroid-mediated development of Arabidopsis

阿拉伯芥膜結合黃體素受體 (Arabidopsis thaliana membrane-associated progesterone receptors, AtMAPRs) 為阿拉伯芥四個具潛在固醇類結合口袋之蛋白質家族，其中位於第二號染色體的AtMAPR2為四個成員中唯一預測不含穿膜區塊者。本實驗室過去對AtMAPR2之相關研究發現，在正常光週期生長條件下，處理高濃度 (uM) 之油菜固醇 (Brassinosteroids) 會抑制AtMAPR2基因之表現。此外， AtMAPR2之基因靜默突變株幼苗對於油菜固醇的處理反應較不敏感。本研究主要探討AtMAPR2對油菜固醇調控之植物生理功能影響，在正常光週期生長條件下，AtMAPR2過度表現突變株幼苗對於低濃度 (nM) 之油菜固醇處理表現出下胚軸及主根延長較顯著之性狀；而AtMAPR2之基因靜默突變株則較不敏感；但在避光生長條件下，突變株間之差異則較不明顯，以上現象符合油菜固醇影響之植物幼苗階段發育與光照之關聯。在發育中晚期時，本研究亦發現AtMAPR2之過度表現突變株呈現較早開花與主莖延長等性狀；而AtMAPR2之基因靜默突變株則呈現較晚開花與主莖較短之性狀。根據以上觀察，推測AtMAPR2於油菜固醇所調控之植物發育與生理扮演一正向調控的角色。在油菜固醇對AtMAPR2之分子層次影響探討中，發現處理油菜固醇之野生型阿拉伯芥幼苗在接受光照後會導致AtMAPR2蛋白質大量累積，但在黑暗環境下並無此現象。在轉錄層次上，處理油菜固醇後幼苗是否接受光照對於AtMAPR2之基因表現並顯著的影響，這代表光照可能透過影響AtMAPR2蛋白質之後轉譯層次調控機制影響其穩定度或功能，進而影響於AtMAPR2相關突變株中觀察到之性狀。Arabidopsis thaliana membrane-associated progesterone receptors (AtMAPRs) share the conserved putative cytochrome b5-like heme/steroid-binding pockets. Among the four members, AtMAPR2 which locates on the second chromosome is the only member without a predicted transmembrane domain. We found that application of micromolar level concentration of BR would suppress the expression of AtMAPR2 under normal photoperiod. Furthermore, the gene silencing mutant of AtMAPR2 seemed insensitive to treatment of BR. In this thesis, a few evidences that linked AtMAPR2’s physiological roles to BR and plant’s responses to light were observed. First, transgenic plant of Arabidopsis that overexpress AtMAPR2 (AtMAPR2-OX) showed an increased elongation in hypocotyls and primary roots in response to treatment of nanomolar level concentration of BR in light. On the contrary, the antisense transgenic plant AtMAPR2-RNAi appeared insensitive to these BR-mediated responses. Furthermore, these differences were not observed when these mutants were grown in dark. Second, AtMAPR2-OX showed an early flowering phenotype and possessed an elongated primary shoot, while the AtMAPR2-RNAi showed late flowering and shorter primary shoot. These results suggested that AtMAPR2 might play a positive role in BR-mediated responses. The protein levels of AtMAPR2 in wild type under BR treatments were monitored using western blot. AtMAPR2 accumulated in the BR-treated Arabidopsis seedlings under light but not under dark, suggesting that illumination might affect the stability of AtMAPR2 via post-translational regulation.口試委員會審定書 i 謝誌 ii Abbreviations iii 摘要 v Abstract vi Chapter 1 Introduction 1 1.1 Brassinosteroids 1 1.1.1 Biosynthesis of BRs 1 1.1.2 Physiological effects of BRs 2 1.1.3 Signal transduction of BRs 3 1.2 Membrane-Associated Progesterone Receptor (MAPR) family 6 1.2.1 Cytochrome b5 like ligand binding domain 7 1.2.2 Putative ligands of MAPR family 7 1.2.3 Putative physiological roles of MAPR family 9 1.3 Arabidopsis thaliana MAPRs (AtMAPRs) 10 1.3.1 Function of AtMAPR5/MSBP1 11 1.3.2 Previous studies on AtMAPR2 12 1.4 Aims of this study 13 Chapter 2 Materials and Methods 15 2.1 Experimental Materials 15 2.1.1 Plant Materials 15 2.2 Chemicals 16 2.2.1 Brassinolide 16 2.3 Instruments 16 2.4 Experimental Methods 18 2.4.1 Culture of Arabidopsis thaliana 18 2.4.2 Analysis of transcription 21 2.4.3 Analysis of translation 28 2.4.4 In silico analysis of protein 38 2.4.5 Observation and analysis of phenotype 40 Chapter 3 Results and Discussion 41 3.1 AtMAPR2-related mutants for phenotypic analysis 41 3.1.1 Selection of homozygous mutants by antibiotic screening 42 3.1.2 Quantitative real-time PCR confirmed the predicted expression level of AtMAPR2 in transgenic mutant lines (AtMAPR2-OX-1-1, AtMAPR2-RNAi-4-2) 43 3.1.3 Protein levels of AtMAPR2 in the mutants 44 3.2 AtMAPR2 might play roles in BR-mediated responses 47 3.2.1 AtMAPR2 might promote BR-mediated elongation of hypocotyl and primary root under illumination 47 3.2.2 AtMAPR2 might participate in shoot elongation and early flowering 51 3.2.3 In silico sequence analysis revealed interesting features of AtMAPR2 53 Chapter 4 Conclusions and Perspectives 57 4.1 Conclusions 57 4.2 Perspectives 58 4.2.1 Observation of other BR-mediated phenotypes 58 4.2.2 Confirmation of BR-mediated alterations in AtMAPR2 under illumination 59 4.2.3 Analysis of the roles that AtMAPR2 plays in BR-mediated development 59 References 61 Figures, Tables, and Scheme 72 Appendices 101 問答集 10