SMAX1/SMXL2 regulate root and root hair development downstream of KAI2-mediated signalling in Arabidopsis

Author summary Karrikins are plant signaling compounds from smoke, which induce germination of fire-following plants. They likely mimic endogenous plant hormones (KAI2-ligands, KLs), because Arabidopsis karrikin receptor mutants display shoot developmental phenotypes. Perception of karrikins/KLs is very similar to that of another plant hormone class, the strigolactones (SLs). Both hormones bind to the related alpha/beta-fold hydrolase receptors KAI2 and D14 respectively, which both interact with the F-box protein MORE AXILLIARY BRANCHES2 (MAX2), for ubiquitylation and subsequent degradation of KL- or SL-signalling specific repressor proteins of the SMXL family. Based on max2 mutant phenotypes it has been suggested that the development of Arabidopsis root architecture and root hairs is regulated by SL signaling. However, max2 does not distinguish between the two signalling pathways. We genetically dissected the role of KL and SL signalling in root and root hair development in Arabidopsis seedlings and show that most root traits are regulated by KL and not by SL signaling: lateral root density is controlled by KL and SL signalling together, while root growth direction, root straightness and root hair development are determined by KL signalling alone. Thus, KL signaling regulates vital plant traits for nutrient and water uptake as well as anchorage to the ground. Karrikins are smoke-derived compounds presumed to mimic endogenous signalling molecules (KAI2-ligand, KL), whose signalling pathway is closely related to that of strigolactones (SLs), important regulators of plant development. Both karrikins/KLs and SLs are perceived by closely related alpha/beta hydrolase receptors (KAI2 and D14 respectively), and signalling through both receptors requires the F-box protein MAX2. Furthermore, both pathways trigger proteasome-mediated degradation of related SMAX1-LIKE (SMXL) proteins, to influence development. It has previously been suggested in multiple studies that SLs are important regulators of root and root hair development in Arabidopsis, but these conclusions are based on phenotypes observed in the non-specific max2 mutants and by use of racemic-GR24, a mixture of stereoisomers that activates both D14 and KAI2 signalling pathways. Here, we demonstrate that the majority of the effects on Arabidopsis root development previously attributed to SL signalling are actually mediated by the KAI2 signalling pathway. Using mutants defective in SL or KL synthesis and/or perception, we show that KAI2-mediated signalling alone regulates root hair density and root hair length as well as root skewing, straightness and diameter, while both KAI2 and D14 pathways regulate lateral root density and epidermal cell length. We test the key hypothesis that KAI2 signals by a non-canonical receptor-target mechanism in the context of root development. Our results provide no evidence for this, and we instead show that all effects of KAI2 in the root can be explained by canonical SMAX1/SMXL2 activity. However, we do find evidence for non-canonical GR24 ligand-receptor interactions in D14/KAI2-mediated root hair development. Overall, our results demonstrate that the KAI2 signalling pathway is an important new regulator of root hair and root development in Arabidopsis and lays an important basis for research into a molecular understanding of how very similar and partially overlapping hormone signalling pathways regulate different phenotypic outputs.