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

环境中的化学物质和植物自身产生的化学物质对植物的生长发育有着深远的影响。这项研究的目的是确定植物是如何感知烟雾中被称为“卡瑞金斯”的一类化学物质的。卡瑞金斯影响重要的农艺性状，如发芽、幼苗生长和抗逆性。已经发现了几个参与卡瑞金感知和反应的基因，但越来越多的证据表明，卡瑞金必须在植物体内转化为活跃信号才能被识别。该项目将确定执行卡里金活化的基因。很可能这些相同的基因参与了一种未知的植物激素的产生，这种激素被卡瑞金斯模仿。这些基因的发现将是了解植物在火灾后如何生长的重要基石，也将是发现一类新的植物生长调节剂的重要基石，这可能为农业改良提供新的机会。该项目使用尖端技术鉴定可能调节卡瑞金代谢的蛋白质复合物成员，然后使用高通量基因编辑方法测试候选基因。该项目将通过为一名博士后研究员、两名研究生和150多名本科生提供培训来增强美国的科学劳动力，其中许多人将是少数民族、来自低收入家庭和/或第一代大学生。将举办每月一次的公众外展活动，向当地社区教授植物科学和STEM学科。这个项目的发现将通过出版物、新闻稿和社交媒体向公众传播。KARRIKIN INSENSITIVE2 （KAI2）是植物中已知的karrikins （KARs）受体和一种未知的内源性KAI2配体（KL）。然而，最近的观察表明，KAI2不能直接识别KARs， KARs必须首先代谢成生物活性信号。KARRIKIN上调的F-BOX1 （KUF1）是KAR信号的转录标记物，其突变导致与KAI2信号过度活跃一致的表型，也导致对KAR1的过敏反应，但对其他KAI2激动剂没有反应。KUF1可能在蛋白质水解依赖的负反馈回路中起作用，调节KL生物合成和KAR1代谢。这一假设将通过研究KUF1如何影响kar1诱导的KAI2靶点SMAX1的降解来验证；kar特异性、kl不敏感拟南芥转基因品系的生长响应以及脉冲处理植物中KAR1的消失率。为了确定KUF1的功能和调控，将进行一系列互补的生化和遗传实验。主要目的是鉴定KUF1多泛素化和降解的靶蛋白，以及确定KUF1蛋白复合物的组成和动力学。亲和纯化-质谱法和酵母双杂交筛选将是鉴定一组潜在的KUF1相互作用物的主要方法，这些相互作用物将通过哺乳动物细胞和植物的生化分析进行验证。将通过高通量crispr - cas9介导的反向遗传筛选来研究编码候选相互作用物的基因，以鉴定kai2依赖性信号活性的kuf1抑制因子和调节因子。假定的KUF1靶点将在植物中检测KUF1依赖的多泛素化和降解。该奖项由综合有机体系统部的生理机制和生物力学项目以及分子和细胞生物科学部的细胞动力学和功能组共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.