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本身产生的化学物质可以对植物的生长和发育产生深远的影响。这项研究的目标是确定植物如何感知烟雾中发现的一种被称为卡里金的化学物质。卡瑞金影响重要的农艺性状，如种子萌发、幼苗生长和抗逆性。已经发现了几个与karrikin感知和反应有关的基因，但越来越多的证据表明，karrikin必须在植物体内转化为活跃的信号才能被识别。该项目将识别执行卡瑞金激活的基因。这些基因很可能参与了一种未知植物荷尔蒙的产生，卡里金模仿这种荷尔蒙。这些基因的发现将是了解植物在火灾后如何生长的重要踏脚石，以及寻找一类可能为农业改良提供新机会的新型植物生长调节剂。该项目使用尖端技术来识别可能调节卡里金新陈代谢的蛋白质复合体成员，然后用高通量的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加强美国的科学队伍，其中许多本科生将是来自低收入家庭的少数族裔和/或第一代大学生。将举行每月一次的公众宣传活动，向当地社区传授植物科学和STEM学科。该项目的发现将通过出版物、新闻稿和社交媒体向公众公布。KARRIKIN INSENSITIVE2(KAI2)是植物中可能的KARS受体(KARS)和一个未知的内源性KAI2配体(KL)。然而，最近的观察表明，KAI2不直接识别KARS，KAI2必须首先被代谢成生物活性信号。KAR信号的转录标记物Karrikin上调的F-BOX1(KUF1)的突变导致与KAI2信号高活性一致的表型，也引起对KAR1的超敏反应，但不引起其他KAI2激动剂的反应。KUF1可能参与调节KL生物合成和KAR1代谢的蛋白水解性负反馈环。这一假设将通过检验KUF1如何影响KAR1诱导的KAI2靶标SMAX1的降解；KAR特异的、KL不敏感的拟南芥转基因株系的生长反应；以及KAR1从脉冲处理的植物中消失的速率来检验。为了确定KUF1的功能和调控方式，将进行一系列补充的生化和遗传实验。主要目标是确定KUF1针对多泛素化和降解的蛋白质，以及定义KUF1蛋白质复合体的组成和动力学。亲和纯化-质谱学和酵母双杂交筛选将是鉴定一组潜在的KUF1相互作用的主要方法，这些相互作用将通过哺乳动物细胞和植物的生化分析进行验证。编码候选相互作用蛋白的基因将通过高通量CRISPR-Cas9介导的反向遗传筛选进行研究，以确定KAI2依赖的信号活性的kuf1抑制子和调节子。假定的KUF1靶标将在植物中测试依赖于KUF1的多泛素化和降解。该奖项是由综合组织系统部门的生理机制和生物力学项目以及分子和细胞生物科学部门的细胞动力学和功能集群共同资助的。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.