New fluorescence sensor system: visualizing activation of CDPKs in real-time.
Plants respond to pathogen attack, drought, nutrient deficiency, and many other challenges with a pronounced change in their intracellular calcium concentrations. This early stress signal activates calcium-dependent protein kinases (CDPKs), which initiate defense cascades by phosphorylating their target proteins, leading to rapid adaptation to the changing environmental conditions. In this process, depending on the stimulus and type of stress, CDPKs coordinate different enzymes in different signaling pathways, thus enabling the plant to specifically respond to the prevailing stress in each case. One of the most exciting questions in this field is, which molecular mechanisms differentially activate the members of the CDPK family to initiate stress-specific signaling cascades. Similarly interesting is the question of how the enzymes are inactivated after the problem has been resolved or the stress has subsided.
IPB scientists, together with partners from Germany and America, have recently shed light on these topics. The first step for the CDPK kinase activity is a conformational change of the enzyme caused by calcium (Ca2+) binding. In their study, the scientists developed a fluorescence-based reporter to visualize Ca2+-dependent conformational changes in CDPK during its activation. To do this, they fitted two Arabidopsis CDPKs (AtCPK21 and AtCPK23), one highly sensitive to Ca2+ and one less sensitive, with the reporter.
After transforming tobacco and Arabidopsis plants with the two CDPK fluorescence constructs, the scientists were able to detect in real-time the calcium-induced fluorescence signal changes in these two plants and thus to follow in vivo the conformational change, i.e. the activation and, after Ca2+ signal decline, also the inactivation of the CDPKs. In Arabidopsis, they focused on the guard cells of the stomata, whereas in tobacco, they mainly observed the pollen tubes, in which a strong fluctuation of the Ca2+ signal naturally occurs. Here, accordingly, only the highly Ca2+-sensitive AtCPK21 fluorescence sensor responded in an oscillatory manner to the intracellular calcium fluctuation, while the less sensitive AtCPK23 fluorescence sensor showed no changes in the fluorescence signal. In Arabidopsis stomatal guard cells, the scientists also demonstrated that the AtCPK21 fluorescence sensor responded to early calcium surges triggered by abscisic acid or by the flagellin peptide flg22. This suggests the involvement of AtCPK21 in pathogen defense.
With this study, the Halle scientists have demonstrated that CDPK isoforms respond differently to calcium signals, which in tissues with pronounced calcium fluctuation leads to the activation of only the highly sensitive CDPKs and thus influences the direction of the signal cascade. In addition, for Arabidopsis guard cells, they were able to make initial statements about the type of stress that had triggered AtCPK21 activation. Therefore, the CDPK fluorescence sensor system is a powerful tool for deciphering Ca2+ signaling pathways in living cells and in real-time in a variety of plant stress and developmental responses, the scientists concluded. The CDPK fluorescence sensor system, named CPKaleon, will be used in the future to elucidate the function of additional CDPK isoforms in various signaling pathways.
