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Karrikin (KAR) molecules found in smoke stimulate seed germination of many plant species that emerge after fire. Genetic studies in Arabidopsis thaliana have identified core components of the KAR signaling pathway, including an α/β-hydrolase, KARRIKIN INSENSITIVE2 (KAI2), that is required for KAR responses. Although KAI2 is often considered a KAR receptor, recent evidence suggests that KARs may require metabolism to become bioactive signals. In addition to sensing KARs or a KAR-derived signal, KAI2 is thought to recognize an unknown endogenous signal, KAI2 ligand (KL). We generated loss-of-function mutations in KARRIKIN-UP-REGULATED F-BOX1 ( KUF1 ), which is a transcriptional marker of KAR/KL signaling in A. thaliana and other plants. The kuf1 mutant in Arabidopsis shows several phenotypes that are consistent with enhanced activity of the KAI2 pathway, including reduced hypocotyl elongation, enhanced cotyledon expansion in light-grown seedlings, increased root hair density and elongation, and differential expression of KAR/KL-responsive transcriptional markers. Seedling phenotypes of kuf1 are dependent on KAI2 and its signaling partner MORE AXILLARY GROWTH2 (MAX2). Furthermore, kuf1 mutants are hypersensitive to KAR 1 , but not to other molecules that can signal through KAI2 such as GR24. This implies that kuf1 does not increase the overall responsiveness of the KAI2-dependent signaling pathway, but specifically affects the ability of KAI2 to detect certain signals. We hypothesize that KUF1 imposes feedback inhibition of KL biosynthesis and KAR 1 metabolism. As an F-box protein, KUF1 likely participates in an E3 ubiquitin ligase complex that imposes this regulation through polyubiquitylation of a protein target(s). 

Abstract KAI2 proteins are plant α/β hydrolase receptors which perceive smoke-derived butenolide signals and endogenous, yet unidentified KAI2-ligands (KLs). The number of functional KAI2 receptors varies among species and KAI2 gene duplication and sub-functionalization likely plays an adaptative role by altering specificity towards different KLs. Legumes represent one of the largest families of flowering plants and contain many agronomic crops. Prior to their diversification, KAI2 underwent duplication resulting in KAI2A and KAI2B. Here we demonstrate thatPisum sativumKAI2A and KAI2B are active receptors and enzymes with divergent ligand stereoselectivity. KAI2B has a higher affinity for and hydrolyses a broader range of substrates including strigolactone-like stereoisomers. We determine the crystal structures of PsKAI2B in apo and butenolide-bound states. The biochemical, structural, and mass spectra analyses of KAI2s reveal a transient intermediate on the catalytic serine and a stable adduct on the catalytic histidine, confirming its role as a bona fide enzyme. Our work uncovers the stereoselectivity of ligand perception and catalysis by diverged KAI2 receptors and proposes adaptive sensitivity to KAR/KL and strigolactones by KAI2B.