Model-driven discovery of calcium-related protein-phosphatase inhibition in plant guard cell signaling

The plant hormone abscisic acid (ABA) promotes stomatal closure via multifarious cellular signaling cascades. Our previous comprehensive reconstruction of the stomatal closure network resulted in an 81-node network with 153 edges. Discrete dynamic modeling utilizing this network reproduced over 75% of experimental observations but a few experimentally supported results were not recapitulated. Here we identify predictions that improve the agreement between model and experiment. We performed dynamics-preserving network reduction, resulting in a condensed 49 node and 113 edge stomatal closure network that preserved all dynamics-determining network motifs and reproduced the predictions of the original model. We then utilized the reduced network to explore cases in which experimental activation of internal nodes in the absence of ABA elicited stomatal closure in wet bench experiments, but not in our in silico model. Our simulations revealed that addition of a single edge, which allows indirect inhibition of any one of three PP2C protein phosphatases (ABI2, PP2CA, HAB1) by cytosolic Ca²⁺ elevation, resolves the majority of the discrepancies. Consistent with this hypothesis, we experimentally show that Ca²⁺ application to cellular lysates at physiological concentrations inhibits PP2C activity. The model augmented with this new edge provides new insights into the role of cytosolic Ca²⁺ oscillations in stomatal closure, revealing a mutual reinforcement between repeated increases in cytosolic Ca²⁺ concentration and a self-sustaining feedback circuit inside the signaling network. These results illustrate how iteration between model and experiment can improve predictions of highly complex cellular dynamics.