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 represents an important mechanism to prevent self-fertilization. This involves interaction of pistil S-locus determinants with a pollen receptor in an incompatible interaction, resulting in a Ca(2+)-dependent signaling network involving activation of a MAPK, p56, and stimulation of several caspase-like activities, resulting in programmed cell death (PCD). MAPK inhibitors provide a useful tool to dissect these mechanisms and distinguish their regulation by different signaling pathways. U0126 is a potent, noncompetitive, and specific inhibitor of MAPK signaling pathways that result in the inhibition of MAPK activation. Here, we describe the use of this drug in combination with a TEY (threonine-glutamic acid-tyrosine) antibody to alter and monitor MAPK activation, together with a range of markers for PCD to implicate a role for MAPK activation in signaling to PCD in pollen tubes. These techniques may be potentially adapted for use in other plant tissues to investigate MAPK activation in other physiologically relevant systems.