Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress

Plant growth is severely affected by hyper-osmotic salt conditions. Althoug h a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentration s (above 125 mm NaCl). In contrast, at high salt concentrations (above 750 mm NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activa ted. By biochemical and immunological analysis, the 46 kDa kinase was ident ified as SIMK, a member of the family of MAPKs (mitogen-activated protein k inases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osm otic conditions. Salt stress induces rapid but transient activation of SIMK , showing maximal activity between 8 and 16 min before slow inactivation. W hen inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nu clear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not corre lated with changes in its intra-cellular compartmentation, suggesting an in tra-nuclear mechanism for the regulation of SIMK activity.