Modulating and Monitoring MAPK Activity During Programmed Cell Death in Pollen
Signal transduction through mitogen-activated protein kinase (MAPK) cascades regulates many cellular responses. One example of a stimulus-mediated MAPK signaling network in plants is the self-incompatibility (SI) response in Papaver rhoeas, which represen
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Introduction Signal transduction through mitogen-activated protein kinase (MAPK) cascades regulates many cellular responses. MAPKs trigger diverse signaling cascades in response to a variety of signals and stimuli (1–6). Experimental evidence has demonstrated the involvement of MAPKs in activation of defense responses, resulting in programmed cell death (PCD) and resistance to pathogens (3–6). The challenge in this area is to assign these modules to specific signaling pathways with defined functions.
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_9, © Springer Science+Business Media, LLC 2011
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S. Li and V.E. Franklin-Tong
One example of a stimulus-mediated MAPK signaling network in plants is the self-incompatibility (SI) response in the field poppy (Papaver rhoeas), which is an important mechanism to prevent inbreeding through specific recognition and rejection of incompatible (“self ”) pollen. SI in Papaver rhoeas can be induced by the addition of recombinant pistil S-proteins (PrsS, Papaver rhoeas stigma S-proteins) (7) to pollen tubes growing in vitro, allowing analysis of events triggered specifically in incompatible pollen. The SI interaction triggers a Ca2+-dependent signaling network, involving rapid increases in cytosolic free Ca2+ (8), depolymerization of F-actin and microtubules (9–11), Ca2+-dependent hyperphosphorylation of two soluble inorganic pyrophosphatases (12) and consequent rapid arrest of pollen tube growth, activation of a p56-MAPK (13, 14), cytochrome c leakage into the cytosol, activation of several caspase-like activities ultimately resulting in cellular dismantling and DNA fragmentation (15, 16). Activation of p56-MAPK was shown to peak at 10 min after SI induction (13), which is after initial arrest of pollen tube growth, indicating that it might be involved in later events triggered by SI. MAPK cascades consist of three kinase modules (MAP kinase kinase kinase: MAP3K/MEKK, MAP kinase kinase: MAPKK/ MAP2K/MEK/MKK, and MAP kinase: MAPK/MPK), which are linked to upstream receptors and downstream targets. MAPKs are highly conserved Ser/Thr kinases across eukaryotes that are activated by dual phosphorylation of Thr and Tyr residues in a TEY/ TDY (threonine–glutamic acid/aspartic acid–tyrosine) motif via specific upstream MAPK kinases (MAP2K/MAP3K). A commercially available, anti-active (-phosphorylated) MAPK pTEpY/ pTDpY antibody (p: pohosphorylated residue) recognizes dually phosphorylated MAPKs and hence allows monitoring of MAPK activity by Western blotting (17, 18). Cell-permeable MAPK pathway inhibitors serve as a powerful tool to dissect MAPK-mediated signaling pathways. U0126 is a chemically synthesized organic compound and a highly potent inhibitor of MAPK cascades (19, 20). Unlike PD098059 that only inhibits activation of inactive MAPKs, U0126 inhibits both active and inactive MAPKs (21, 22). U0126 has been widely used to block MAPK cascades and assign them to specific signa
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