In Lima bean, the first response to herbivore attack is a strong Vm depolarization in the bite zone, followed by a transient Vm hyperpolarization and, finally, a constant Vm depolarization throughout the rest of the attacked leaf. Continuous recording of the Vm during herbivore feeding show rapid Vm changes followed by constant Vm depolarizations (top left). In mechanically damaged Lima bean tissues, application of exogenous increasing H2O2 concentrations trigger a transient Vm depolarization that can be recovered by washing tissues (top right).Using the membrane-permeant Ca2+-selective fluorescent dye, Fluo-3 AM, the [Ca2+]cyt can be determined by confocal laser scanning microscopy in herbivore-wounded leaf tissue. In the bite zone, there is a dramatic Ca2+ influx limited to few cell layers (centre left). H2O2 can trigger a Ca2+ response in transgenic soybean suspension cells expressing the Ca2+-sensitive aequorin system. The Ca2+response was determined in a concentration-dependent fashion, and the transiently accumulating [Ca2+]cyt is linearly correlated with the amount of H2O2 up to 0.1mM (bottom left). 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) can be used to detect the presence of active peroxidases and the release of H2O2 from biological samples, with particular reference to mitochondria. The subcellular localization of H2O2 production in herbivore wounded leaves following incubation with Amplex Red shows a clear localization in mitochondria/peroxisomes (stained in yellow, single arrow) and adjacent to the plasma membrane (double arrow). Chloroplasts in the bottom right figure are stained in blue. From Maffei et al., Plant Physiology (2006). The use of this picture requires the written permission from M. Maffei.