Strigolactones (SLs) are a recently discovered class of endogenous plant hormones that regulate many integral traits such as leaf senescence and root elongation while also functioning as exuded signaling molecules. In pea (Pisum sativum), key players of this hormone pathway include RMS3 (an α/β-fold hydrolase) and PsMAX2 (the F-box component of an SCF E3 ubiquitin ligase module) that coordinate SL-dependent degradation of transcriptional repressors. Strikingly, RMS3 functions as both the SL receptor and an active serine hydrolase that hydrolyzes SLs after they are perceived. This characteristic has raised the questions of whether SL hydrolysis or its mere perception is required for signal propagation, and how exactly the coordination of PsMAX2-RMS3 is related to SL hydrolysis. Here, we elucidate the first crystal structure of RMS3, investigate its enzymatic function by generating two catalytic mutants, and study their effects in SL signaling in vitro. The data uncovered in this work hold the potential to broadly impact research into SL perception and signaling in legumes and beyond.

Plant signaling pathways control all aspects of plant physiology and development, providing plants with the flexibility to respond to both internal and external cues. In recent years there has been a growing body of research dedicated to plant phytohormones and understanding the interplay between hormone signaling pathways. In my research work, I explored the karrikin (KAR) signaling pathway, a small molecule-induced network initially found to be responsive to smoke, but now recognized for its diverse effects on plant phenotypes in the absence of fire. In this thesis and elsewhere, there is substantial evidence that KAR signaling is a novel hormone signaling network. In this PhD research I have pushed the boundaries of knowledge in this KAR signaling field.In the first chapter I introduce the critical functions of the KAR signaling pathway and its intricate relation with the phytohormone strigolactone (SL). I delve into the intriguing story of how KAR signaling came to be known as ‘KL’, an undiscovered endogenous signaling pathway with broad implications for plant growth and development. The second chapter presents the results of a study investigating the effect of duplicating the KAR receptor, KAI2 across evolutionary time. This chapter illustrates how receptors can fine-tune their sensitivity to different ligands by modifying just a few amino acids in the ligand-binding pocket. Additionally, I reveal a novel ligand-bound intermediate structure of KAI2, shedding light on the complex mode of ligand interaction and hydrolysis. In the third chapter I explore mutational studies of the KAI2 receptor, examining again its ligand binding pocket. I show how one mutation can enhance sensitivity while another diminishes it. I also examine perturbation to the protein-protein interaction interface of KAI2 with its binding partner, MAX2, revealing residues and hydrophobic interactions that contribute to this interaction. In the fourth chapter I investigate a novel KL discovered in petunia, which exerts broad effects on floral development. I untangle the intricate relations between this novel KL, its ability to induce a KL molecular response, and its reliance on the KAR/KL signaling pathway to do so. In the fifth chapter I reveal the dynamics within the KL signaling protein MAX2, demonstrating how conformational changes affect its ability to mediate ubiquitination and degradation of target substrates, as well as its impact on the activity of the receptor protein binding partner. In the sixth and final chapter I introduce a new area of research that I have pioneered, exploring the interactome of the KL signaling pathway in plants. Here I offer insights into potential novel proteins involved in the KL signaling pathway, as well as those that regulate or engage in cross-talk between KL and other hormone signaling pathways.