Identification of Novel Inhibitors of Auxin-Induced Ca²⁺ Signaling via a Plant-Based Chemical Screen

Many signal perception mechanisms are connected to Ca²⁺-based second messenger signaling to modulate specific cellular responses. The well-characterized plant hormone auxin elicits a very rapid Ca²⁺ signal. However, the cellular targets of auxin-induced Ca²⁺ are largely unknown. Here, we screened a biologically annotated chemical library for inhibitors of auxin-induced Ca²⁺ entry in plant cell suspensions to better understand the molecular mechanism of auxin-induced Ca²⁺ and to explore the physiological relevance of Ca²⁺ in auxin signal transduction. Using this approach, we defined a set of diverse, small molecules that interfere with auxin-induced Ca²⁺ entry. Based on annotated biological activities of the hit molecules, we found that auxin-induced Ca²⁺ signaling is, among others, highly sensitive to disruption of membrane proton gradients and the mammalian Ca²⁺ channel inhibitor bepridil. Whereas protonophores nonselectively inhibited auxin-induced and osmotic stress–induced Ca²⁺ signals, bepridil specifically inhibited auxin-induced Ca²⁺. We found evidence that bepridil severely alters vacuolar morphology and antagonized auxin-induced vacuolar remodeling. Further exploration of this plant-tailored collection of inhibitors will lead to a better understanding of auxin-induced Ca²⁺ entry and its relevance for auxin responses.