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

合作研究：发现植物中控制 karrikin 和 KAI2 配体代谢的负反馈机制

• 批准号: 1856741
• 负责人: David Nelson
• 金额: $ 64.12万
• 依托单位: University of California-Riverside
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-15 至 2023-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1856741&HistoricalAwards=false
• 关键词: Collaborative; Research; Discovery; negative; feedback

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.

环境中的化学物质和植物本身产生的化学物质会对植物的生长和发育产生深远的影响。这项研究的目的是确定植物如何感知烟雾中发现的一类称为卡里金的化学物质。 Karrikins 影响重要的农艺性状，例如发芽、幼苗生长和胁迫耐受性。已经发现了一些与 karrikin 感知和反应有关的基因，但越来越多的证据表明，karrikin 必须在植物内转化为主动信号才能被识别。该项目将鉴定激活 karrikins 的基因。这些相同的基因很可能参与了卡里金模仿的未知植物激素的产生。这些基因的发现将成为了解植物在火灾后如何生长以及寻找可能为农业改良提供新机会的新型植物生长调节剂的重要基石。该项目使用尖端技术来识别可能调节 karrikin 代谢的蛋白质复合物成员，然后通过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，其中许多人是来自低收入家庭的少数族裔和/或第一代大学生。我们将每月举办一次公共宣传活动，向当地社区传授植物科学和 STEM 学科知识。该项目的发现将通过出版物、新闻稿和社交媒体向公众传达。KARRIKIN INSENSITIVE2 (KAI2) 是植物中 karrikins (KAR) 的推定受体，也是一种迄今未知的内源 KAI2 配体 (KL)。然而，最近的观察表明，KAI2 不能直接识别 KAR，并且 KAR 必须首先代谢成生物活性信号。 KARRIKIN UPREGULATED F-BOX1 (KUF1)（KAR 信号传导的转录标记）中的突变会导致与过度活跃的 KAI2 信号传导一致的表型，并且还会引起对 KAR1 的过敏反应，但不会引起对其他 KAI2 激动剂的过敏反应。 KUF1 可能在蛋白水解依赖性负反馈环路中发挥作用，调节 KL 生物合成和 KAR1 代谢。该假设将通过检查 KUF1 如何影响 KAR1 诱导的 KAI2 靶标 SMAX1 降解来检验； KAR 特异性、KL 不敏感拟南芥转基因品系的生长反应；以及脉冲处理植物中 KAR1 消失的速度。为了确定 KUF1 的功能和调节方式，将进行一系列补充性的生化和遗传实验。主要目标是鉴定 KUF1 进行多泛素化和降解的目标蛋白质，以及定义 KUF1 蛋白质复合物的成分和动力学。亲和纯化-质谱和酵母双杂交筛选将是鉴定一组潜在 KUF1 相互作用子的主要方法，这些相互作用子将通过哺乳动物细胞和植物中的生化测定进行验证。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子的基因，以鉴定 KAI2 依赖性信号传导活性的 kuf1 抑制子和调节子。假定的 KUF1 靶标将在植物中进行 KUF1 依赖性多泛素化和降解测试。该奖项由综合有机体系统部门的生理机制和生物力学计划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。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

A KARRIKIN INSENSITIVE2 paralog in lettuce mediates highly sensitive germination responses to karrikinolide

• DOI: 10.1101/2021.10.13.464162
• 发表时间: 2021-10
• 期刊: bioRxiv
• 作者: Stephanie E. Martinez;Caitlin E. Conn;Angelica M. Guercio;C. Sepulveda;Christopher J. Fiscus;Daniel Koenig;N. Shabek;David C. Nelson

Desmethyl butenolides are optimal ligands for karrikin receptor proteins

去甲基丁烯内酯是 karrikin 受体蛋白的最佳配体

• DOI: 10.1111/nph.17224
• 发表时间: 2021
• 期刊: New Phytologist
• 影响因子: 9.4
• 作者: Yao, Jiaren;Scaffidi, Adrian;Meng, Yongjie;Melville, Kim T.;Komatsu, Aino;Khosla, Aashima;Nelson, David C.;Kyozuka, Junko;Flematti, Gavin R.;Waters, Mark T.
• 通讯作者: Waters, Mark T.

The mechanism of host-induced germination in root parasitic plants

• DOI: 10.1093/plphys/kiab043
• 发表时间: 2021-02-03
• 期刊: PLANT PHYSIOLOGY
• 影响因子: 7.4
• 作者: Nelson, David C.

A Course-Based Undergraduate Research Experience for High-Throughput Reverse Genetic Studies in Arabidopsis Thaliana with CRISPR-Cas9

• DOI: 10.20944/preprints202008.0619.v1
• 发表时间: 2020-08
• 作者: Alison M. Mills;V. Jagannatha;Alejandro Cortez;Michael A Guzmán;J. Burnette;Matthew A. Collin