High-resolution imaging of Ca2+, redox status, ROS and pH using GFP biosensors

Many plant response systems are linked to complex dynamics in signaling molecules such as Ca2+ and reactive oxygen species (ROS) and to pH. Regulatory changes in these molecules can occur in the timeframe of seconds and are often limited to specific subcellular locales. Thus, to understand how Ca2+, ROS and pH form part of plants regulatory networks, it is essential to capture their rapid dynamics with resolutions that span the whole plant to subcellular dimensions. Defining the spatio-temporal signaling signatures of these regulators at high resolution has now been greatly facilitated by the generation of plants expressing a range of GFP-based bioprobes. For Ca2+ and pH, probes such as the yellow cameleon Ca2+ sensors (principally YC2.1 and 3.6) or the pHluorin and H148D pH sensors provide a robust suite of tools to image changes in these ions. For ROS, the tools are much more limited, with the GFP-based H2O2 sensor Hyper representing a significant advance for the field. However, with this probe, its marked pH sensitivity provides a key challenge to interpretation without using appropriate controls to test for potentially coupled pH-dependent changes. Most of these Ca2+-, ROS- and pH-imaging biosensors are compatible with the standard configurations of confocal microscopes available to many researchers. These probes therefore represent a readily accessible toolkit to monitor cellular signaling. Their use does require appreciation of a minimal set of controls but these are largely related to ensuring that neither the probe itself nor the imaging conditions used perturb the biology of the plant under study.