A Study on the Signaling Pathway of Salt- and Bradyrhizobium japonicum-Activated MAPKs in Soybean

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2013. 2. 안정선.ABSTRACT Soybeans MAPKs were first reported in early 2000s. However, identification and signaling pathway of soybean MAPKs was barely known. Therefore, I studied the signaling pathway and regulation mechanism of a salt-stress activated 47-kD MAPK, GMK1, and Bradyrhizobium japonicum culture filtrate activated two MAPKs, GMK1 and GMK2. Concerning the salt stress, activity of GMK1 increased with increasing salt concentrations up to 300 mM NaCl after 5 min of the treatment and was regulated by post-translational modification. I found that mastoparan, a heteromeric G-protein activator, also activated GMK1, and that n-butanol, a phospholipase D inhibitor, and neomycin, a phospholipase C inhibitor, inhibited its activity. Moreover, GMK1 activity was reduced by suramin, a heteromeric G-protein inhibitor, and by two inhibitors of phosphatidic acid (PA) generation after 5 min of 300 mM NaCl treatment. Endogenous PA levels were highest 5 min after induction of salt stress, and exogenous PA directly activated GMK1. These results suggest salt signaling passed from heteromeric G-protein to GMK1 via PA in early time of the stress. On the other hand, H2O2 also activated GMK1 even in the presence of PA generation inhibitors, but GMK1 activity was greatly decreased in the presence of diphenyleneiodonium, an inhibitor of NADPH-oxidase after 5 min of 300 mM NaCl treatment. On the contrary, the n-butanol and neomycin reduced GMK1 activity within 5 min of the treatment. Thus, GMK1 activity may be sustained by H2O2 10 min after the treatment. In the relationship between GMK1 and ROS generation, ROS generation was reduced by SB202190, a MAPK inhibitor in NaCl treatment. Moreover, ROS was increased in protoplast only overexpressing TESD-GMKK1, a GMK1 activator in vitro, but these effects were occurred at prolonged time of NaCl treatment. These data suggest that GMK1 indirectly regulates ROS generation. Further, GMK1 was translocated into the nucleus 60 min after NaCl treatment. The translocation is reduced by SB202190. Hydrogen peroxide induced translocation of GMK1 to nucleus and DPI disturbed nuclear translocation of GMK1 in salt stress. It is means that translocation of GMK1 by 300 mM NaCl treatment is mediated by H2O2 in soybean. Concerning the interaction between soybean and Bradyrhizobium japonicum, GMK1 and GMK2 were differently regulated by GCF treatment until 15 h of the treatmentGMK1 was continually activated, but activity of GMK2 was reduced after 3 h of the treatment. Moreover, GMK1 is regulated at post-translational level, but GMK2 was regulated at translational level. In the treatment of PA generation inhibitors, activities both GMK1 and GMK2 were reduced. However, in calcium signaling blocking, only activity of GMK2 was reduced. Therefore, these two MAPKs may play different roles in symbiotic interaction. However, the evidence of these MAPKs involvement on Nod factor signaling is not clear. Therefore, role of MAPKs in symbiotic interaction should be more elucidated. Consequently, GMK1 is dually activated by PA and H2O2, and translocated to nucleus mediated by H2O2 in salt stress. Moreover, GCF treatment activated both GMK1 and GMK2, but these MAPKs were differently regulated in many aspects.Contents Abstract………………………………………………………………..………….1 Contents…………………………………………………………………………...3 List of Figures……………………………………………………………………6 Abbreviations……………………………………………………………………….8 Chapter 1. General introduction………………………………………………..10 1-1. Mitogen-activated protein kinase………………………………………...10 1-2. Salt stress……………………………………………………………………..14 1-3. Soybean……………………………………………………………………....18 1-4. Nodulation……………………………………………………………............22 1-5. Objective……………………………………………………………..............23 Chapter 2. Signaling pathway of GMK1 in salt stress…...................................24 2-1. Introduction……………………………………………….………………25 2-1.1. Salt stress signaling pathway.................................................................25 2-1.2. Phosphatidic acid and hydrogen peroxide............................................25 2-1.3. Soybean MAPK.......................................................................................26 2-1.4. Nuclear translocation of MAPK............................................................27 2-2. Materials and methods…………………………………………………..28 2-2.1. Plant material ……………………………………………………..….28 2-2.2. Preparation of protein extracts and western blot analysis………..…28 2-2.3. Immunoprecipitation (IP) and in-gel kinase assay…………………28 2-2.4. Total RNA isolation and northern blot analysis………………….…29 2-2-5. Phospholipid isolation and thin-layer chromatography (TLC)..….29 2-2.6. H2O2 and O2- measurement………………………………………..…30 2-2.7. Protoplast isolation and polyethylene glycol transfection………….30 2-2.8. Immunolocalization assay......................................................................31 2- 2.9. Nuclei isolation.........................................................................................31 2-3. Results………………………………………………………………………31 2-3.1. Salt stress activates GMK1 via post-translational regulation……..31 2-3.2. GMK1 activity is regulated by PA via heteromeric G-protein, PLD, and PLC under salt stress conditions …………….…..........................33 2-3.3. Reactive oxygen species were generated in salt-treated soybean roots………………………………………………………….....34 2-3.4. Hydrogen peroxide activates GMK1 independently of PA…………35 2-3.5. Activity of GMK1 is regulated by PA and hydrogen peroxide at different time points in salt stress…………………………….…….......36 2-3.6. Reactive oxygen species generation is indirectly regulated by GMK1………………………………………………………….…...…36 2-3.7. Total SOD activity in 300 mM NaCl treatment……………….....…..37 2-3.8. Morphological analysis of soybean in salt stress with GMK1 signaling pathway inhibitors…………………………………………………..…..38 2-3.9. GMK1 is translocated to nucleus by NaCl treatment..........................39 2-3.10. Nuclear translocation of GMK1 is reduced by MAPK inhibitor..40 2-3.11. Nuclear translocation of GMK1 is induced by hydrogen peroxide..41 2-3.12. Nuclear translocation of GMK1 is mediated by endogenous hydrogen peroxide in salt stress...............................................................41 2-4. Discussion.......................................................................................................65 Chapter 3. Two soybean mitogen-activated protein kinases are involved in Bradyrhizobium-soybean interaction……....................................76 3-1. Introduction...................................................................................................75 3-1.1. Nitrogen fixation.....................................................................................75 3-1.2. Nod factor signaling................................................................................75 3-1.3. MAPKs.....................................................................................................76 3-2. Materials and methods…………………………………….......…………77 3-2.1. Plant material …………………………………………………………77 3-2.2. Bacterial culture and preparation of culture filtrate…………..77 3-2.3. In-gel kinase assay…………………………………………………….77 3-2.4. Immunoprecipitaion…………………………………………………..78 3-2.5. Total RNA isolation and RT-PCR…………………………………….78 3-2.6. Preparation of protein extracts and western blot analysis………......79 3-3. Results.............................................................................................................79 3-3.1. Two soybean MAPKs, GMK1 and GMK2 are activated by GCF treatment…………………………………………………………..80 3-3.2. GMK2 activity is regulated at translational level……………….….80 3-3.3. GMK1 and GMK2 are regulated by PA and GMK2 is regulated by calcium in GCF treatment………………………………………….80 3-4. Discussion.......................................................................................................86 References..............................................................................................................89 Conclusion............................................................................................................104 국문초록…………………………………………………………………………106Docto