Novel Mechanisms of Strigolactone-Induced DWARF14 Degradation in Arabidopsis thaliana

Strigolactones (SLs) are carotenoid-derived compounds that regulate various aspects of plant development, physiological responses and plant interactions with microorganisms. In angiosperms, the SL receptor is the /{beta} hydrolase D14 that, upon SL binding, undergoes conformational changes, triggers SL-dependent responses and hydrolyses SLs. Arabidopsis SL signalling involves the formation of a complex between SL-bound D14, the E3-ubiquitin ligase SCFMAX2 and the transcriptional corepressors SMXL6/7/8 (SMXLs), which become ubiquitinated and degraded by the proteasome. However, the sequence of events that result in SL signalling and the requirement of SL hydrolysis for this process remain unclear. In addition, SL destabilises the D14 receptor. The biological significance of SL-induced D14 degradation is unclear, although it has been proposed to create a negative feedback loop in SL signalling. The current model proposes that D14 degradation occurs after SMXLs ubiquitination and proteolysis, via the same E3-ubiquitin ligase that targets the repressors. In this work we quantitatively studied the degradation dynamics of Arabidopsis D14 in response to SLs in planta. For this, we conducted fluorescence and luminescence assays to monitor D14 stability dynamics upon SL treatments, in transgenic lines expressing D14 fused to GREEN FLUORESCENT PROTEIN (GFP) or LUCIFERASE (LUC), in wild-type and SL-signalling mutant backgrounds. Mutant D14 proteins predicted to be non-functional for SL signalling were also examined, and their capability to bind SLs in vitro was studied using Differential Scanning Fluorimetry (DSF). Finally, we used a non-hydrolysable SL to test the requirements of SL hydrolysis for D14 and SMXL7 degradation. Our research revealed that SL-induced D14 degradation may occur in the absence of SCFMAX2 and/or SMXLs by a proteasome-independent mechanism. Additionally, we observed conditions in which the efficiency of SL-induced degradation of D14 is not aligned with that of SMXL7 degradation. Finally, our results indicate that the hydrolysis of SLs is not a prerequisite to trigger either D14 or SMXL7 degradation. These findings suggest the existence of a regulatory mechanism governing D14 degradation more complex than anticipated, and provide novel insights into the dynamics of SL signalling in Arabidopsis.