Přehled o publikaci

Cytokinins (CKs) are phytohormones that regulate diverse aspects of plants growth and development. In Arabidopsis thaliana, CKs bind to the extracellular CHASE (cyclases/histidine kinases associated sensory extracellular domain) domain of sensory histidine kinases (AHK2, AHK3 and AHK4) activating multistep phosphorelay (MSP) signaling with the primary response genes regulating CK-dependent plant responses (Hwang, Sheen and Muller 2012). Based on the known structure of CK-binding AHK4 CHASE domain (Hothorn, Dabi and Chory 2011), the in silico screen was performed to search for the ligands that could bind and control the activity of CK receptors leading to either CK-like or anti CK effects upon exogenous plant treatment. Four predicted compounds were assessed for their affinity to CK receptors using a competition binding assay with radioactively labelled naturally occurring CK trans-Zeatin (Romanov et al., 2005). To discriminate between agonistic or antagonistic mode of binding, a reporter system wherein CK receptor activation induces β-galactosidase expression in Escherichia coli was used (Suzuki et al. 2001). Root elongation of Arabidopsis seedlings and chlorophyll dynamics during the initial stages of de-etiolation were examined upon treatment with the predicted ligands. Additionally, transgenic Arabidopsis line carrying TCSn::GFP, a CK-responsive synthetic reporter (Muller and Sheen 2008), were used to evaluate transcriptional activation of CK signaling in a response to exogenous treatment with the predicted ligands. Two ligands (MU2239 and MU2240) showed affinity to AHK3 and AHK4 receptors and activated them in a dose-depended manner. A weak activation of AHK4 was also noted with application of ligand MU2238. The observed root growth inhibition as well as chlorophyll kinetics pattern in seedlings during first four hours of de-etiolation suggests CK-like effect of ligands MU2239 and MU2240. In line with that, exogenous treatment with MU2239 and MU2240 ligands resulted into activation of CK-responsive TCSn::GFP reporter line. Based on that, we conclude CK-like nature of predicted ligands MU2239 and MU2240, providing evidence for successful employment of virtual screening for molecular docking to gain new phytohormones with promising potential in agriculture applications.