Analysis of early wound signal transduction events in plants

Wounding is one of the most severe environmental stresses plants have to cope with. It can be caused by mechanical injury, herbivore or pathogen attack. Plants respond to this threat by an array of events including reversible protein phosphorylation, synthesis of the plant hormone jasmonic acid, changes in gene expression and metabolic adaptation to satisfy the increased energy demand. Recently, we have provided evidence that two novel protein kinases WAG (wound-activated glycogen synthase kinase 3) and WIG (wound-induced glycogen synthase kinase 3) might be involved in the early wound response in plants. In this research project a genetic approach is proposed to place the WAG and WIG kinases on the wound signaling cascade. Arabidopsis plants disrupted in the WAG and/or WIG function will be used (i) to analyse whether WAG and/or WIG kinases regulate wound-induced JA accumulation, (ii) to study how wound-induced changes in gene expression are regulated by p50WAG and p53WIG by microarray and cluster analysis, and (iii) to determine whether MAP kinases and WAG and WIG kinases act on the same or on different pathways. Additionally, interaction partners of WAG and WIG, which have been isolated by a yeast two-hybrid screen, will be further analysed. These clones indicate that WAG and WIG might regulate gene expression by modulating transcription factor activities and might link signal transduction to wound-induced metabolic adaptation.

Plants are permanently exposed to a multitude of environmental signals. These external stimuli have to be perceived at the cellular level and subsequently transduced via signaling cascades to induce a proper cellular response. Reversible protein phosphorylation constitutes a major mechanism of intracellular information transfer. Glycogen synthase kinase 3 (GSK-3) is a serinehreonine protein kinase that plays a key role in many physiological processes. For example, animal GSK-3 is essential for proper cell fate determination, cell cycle regulation, and is involved in apoptosis and cancer. While mammals have two GSK-3 isoforms, plants contain a GSK-3/shaggy-like kinase (GSK) multigene family. Only recently some functional data on plant GSKs became available indicating that distinct GSKs are involved in different physiological processes such as flower development, brassinosteroid signaling, and stress responses. Before we had shown that WIG (wound-induced GSK) and WAG (wound-activated GSK), two GSKs from Medicago sativa, are activated in response to wounding. Signaling is highly complex with a substantial network of regulatory interactions and coordinations. Many key signal transduction components are shared by different pathways. Thus we tested whether WIG and WAG might be involved in other processes than the wound response. Indeed, we could show that WAG is not only involved in the plants response to wounding but might also be a critical component during the high salinity stress response. WAG kinase activity is rapidly induced by exposure of roots to high salt concentrations but not by cold or drought. Arabidopsis plants that constitutively overexpress WAG were significantly more resistant against high salt concentrations than control plants indicating that WAG is a positive regulator of salt stress tolerance. Intriguingly, immunolocalization studies revealed that WAG is a plastid-localized GSK associated with starch grains. Within the framework of this project we tested whether other members of the GSK family could be involved in stress signaling, too. Biochemical analysis revealed MsK1 as stress-responsive GSK. Immunokinase assays with MsK1-specific antibodies showed that MsK1 is active in leaves and roots under normal growth conditions. However, when leaves were treated with the herbicide paraquat, which induces the production of superoxide in chloroplasts, MsK1 kinase activity decreased rapidly. In roots cellulase treatment lead to a rapid downregulation of MsK1 activity. Downregulation of MsK1 was followed by proteasome-dependent protein degradation of MsK1. To analyse the functional role of MsK1 transgenic Arabidopsis plants constitutively overexpressing MsK1 were generated and their response to different stresses were analysed. In germination assays MsK1 overexpression plants displayed enhanced resistance to cellulase and paraquat. Interestingly, MsK1 overexpressing plants were more susceptible to a virulent Pseudomonas syringae bacterial strain but not to an avirulent isolate. Taken together, our work showed that several distinct GSKs are involved in the plants response to different abiotic and biotic stresses. The activity of distinct GSKs appear to be regulated differently. While WAG and WIG activities are low under normal growth conditions and are stimulated by stress MsK1 seems to be constantly active but downregulated upon stress. The proteasome-dependent degradation of MsK1 constitutes a new mode of GSK regulation that had not been described for GSK-3 in any other organism.