Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

Centre of the Region Haná for Biotechnological and Agricultural Research Department of Cell Biology Faculty of Science Palacký University Šlechtitelů 11 783 71 Olomouc Czech Republic

Department of Rickettsiology Institute of Virology Slovak Academy of Sciences Dubravska cesta 9 Bratislava 845 05 Slovakia

Gregor Mendel Institute of Molecular Plant Biology GmbH Dr Bohr Gasse 3 A 1030 Vienna Austria

Institution of Cell Imaging and Ultrastructure Research University of Vienna Althanstrasse 14 A 1090 Vienna Austria

Max F Perutz Laboratories Vienna Biocenter University of Vienna Dr Bohr Gasse 9 A 1030 Vienna Austria

Unité de Recherche en Genomique Végétale Université d'Evry Val d'essone 2 rue Gaston Crémieux F 91057 Evry France

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