Collaborative Research: Discovery of a negative feedback mechanism that controls karrikin and KAI2 ligand metabolism in plants

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Chemicals in the environment and chemicals produced by plants themselves can have profound effects on plant growth and development. The goal of this study is to determine how plants sense a class of chemicals found in smoke known as karrikins. Karrikins influence important agronomic traits such as germination, seedling growth, and stress tolerance. Several genes that are involved in karrikin perception and response have been found, but accumulating evidence suggests that karrikins must be converted into active signals within plants before they can be recognized. This project will identify genes that carry out the activation of karrikins. It is likely that these same genes are involved in production of an unknown plant hormone that karrikins mimic. Discovery of these genes will be a significant steppingstone to understanding how plants grow after fires, as well as to finding a novel class of plant growth regulators that may offer new opportunities for agricultural improvement. This project uses cutting-edge techniques for identifying members of protein complexes that may regulate karrikin metabolism, and then tests candidate genes with a high-throughput gene-editing approach. This project will enhance the U.S. scientific workforce by providing training for one postdoctoral researcher, two graduate students, and over 150 undergraduates, many of whom will be underrepresented minorities, from low-income families, and/or first-generation college students. Monthly public outreach events that teach the local community about plant science and STEM disciplines will be held. Discoveries from this project will be communicated to the public through publications, press releases, and social media.KARRIKIN INSENSITIVE2 (KAI2) is the putative receptor in plants for karrikins (KARs) and an as-yet-unknown endogenous KAI2 ligand (KL). However, recent observations suggest that KAI2 does not recognize KARs directly and that KARs must first be metabolized into a bioactive signal. Mutations in KARRIKIN UPREGULATED F-BOX1 (KUF1), a transcriptional marker of KAR signaling, cause phenotypes that are consistent with hyperactive KAI2 signaling and also cause hypersensitive responses to KAR1, but not other KAI2 agonists. KUF1 may act in a proteolysis-dependent negative feedback loop that regulates KL biosynthesis and KAR1 metabolism. This hypothesis will be tested by examining how KUF1 influences KAR1-induced degradation of the KAI2 target SMAX1; growth responses of a KAR-specific, KL-insensitive Arabidopsis transgenic line; and the rates of KAR1 disappearance from pulse-treated plants. To determine how KUF1 functions and is regulated, a complementary series of biochemical and genetic experiments will be performed. The primary objectives are to identify proteins that are targeted by KUF1 for polyubiquitylation and degradation, as well as define the components and dynamics of the KUF1 protein complex. Affinity purification-mass spectrometry and yeast two-hybrid screens will be primary approaches to identify a set of potential KUF1 interactors that will be validated by biochemical assays in mammalian cells and plants. Genes encoding candidate interactors will be investigated through a high-throughput CRISPR-Cas9-mediated reverse genetic screen to identify kuf1 suppressors and modulators of KAI2-dependent signaling activity. Putative KUF1 targets will be tested for KUF1-dependent polyubiquitylation and degradation in plants. This award was co-funded by the Physiological Mechanisms and Biomechanics Program in the Division of Integrative Organismal Systems and the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.