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

Abstract

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 treatment

GMK1 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.