Signals of different nature are transduced in cells through signal transduction pathways, where mitogen-activated protein kinases (MAPKs) play an important role as signaling molecules. Views into intracellular localization of MAPKs are critical for the understanding of their spatial and temporal functions, like activation-based relocation, compartmentation, or interactions with local substrates. Localization of MAPKs in cells is thus very useful cell biological approach, extending complex mode of cell signaling characterization in plants. Here, we present a method for subcellular immunofluorescence localization of MAPKs using protein- or phospho-specific antibodies, performed on sectioned fixed plant samples. It is based on embedding of samples in the Steedman's wax, a low-melting point polyester wax embedding medium, which maintains high antigenicity of studied proteins. In addition, exposure of dewaxed sections to antibodies allows for their efficient penetration. Altogether, it makes this simple method a good tool in the efficient subcellular localization of diverse proteins, including plant MAPKs.
- MeSH
- Arabidopsis cytologie účinky léků enzymologie MeSH
- fluorescenční mikroskopie MeSH
- fluorescenční protilátková technika metody MeSH
- Medicago sativa cytologie účinky léků enzymologie MeSH
- mitogenem aktivované proteinkinasy analýza metabolismus MeSH
- polyestery * MeSH
- transport proteinů MeSH
- vosky * MeSH
- zalévání tkání metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- aktivace enzymů MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- exprese genu MeSH
- geneticky modifikované rostliny genetika růst a vývoj metabolismus MeSH
- Medicago sativa enzymologie genetika MeSH
- mitogenem aktivované proteinkinasy kinas genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- semenáček genetika růst a vývoj metabolismus MeSH
- soli metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH