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.

植物本身产生的环境中的化学物质和化学物质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在被称为Karrikins的烟雾中发现一类化学物质。 Karrikins影响着重要的农艺性状，例如发芽，幼苗生长和胁迫耐受性。已经发现了与Karrikin感知和反应有关的几个基因，但是积累的证据表明，在识别植物之前，必须将Karrikins转化为植物内的活动信号。该项目将确定执行Karrikins激活的基因。这些相同的基因可能与Karrikins模仿的未知植物激素有关。这些基因的发现将是了解植物在火灾后如何生长的重要垫脚石，以及寻找一种新型的植物生长调节剂，这些调节剂可能为农业改善提供新的机会。该项目使用尖端技术来识别可能调节卡里金代谢的蛋白质复合物的成员，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，从而增强美国科学劳动力，其中许多人将来自低收入家庭的少数群体和/或第一代大学生。将举行每月公共宣传活动，以教授当地社区有关植物科学和STEM学科的信息。该项目的发现将通过出版物，新闻稿和社交媒体传达给公众。Karrikinnosistive2（Kai2）是Karrikins（KARS）植物的推定受体和尚未尚未尚未内源的Kai2 Ligand（KL）。但是，最近的观察结果表明，KAI2并未直接识别KAR，并且必须首先将KARS代谢为生物活性信号。 Karrikin中的突变上调F-box1（KUF1）是KAR信号传导的转录标记，导致表型与过度活跃的KAI2信号一致，并且还会对KAR1产生过度敏感的反应，但没有其他KAI2激动剂。 KUF1可以在调节KL生物合成和KAR1代谢的蛋白水解依赖性反馈回路中起作用。该假设将通过研究KUF1如何影响KAR1诱导的KAI2靶标SMAX1降解来检验。 KAR特异性，不敏感的拟南芥转基因线的生长反应； Kar1的速率消失了经脉搏处理的植物。为了确定KUF1的功能和调节方式，将进行一系列互补的生化和遗传实验。主要目标是鉴定由KUF1靶向多泛素化和降解的蛋白质，并定义KUF1蛋白复合物的成分和动力学。亲和力纯化 - 质谱法和酵母两杂交筛网将是识别一组潜在的KUF1相互作用的主要方法，这些方法将通过哺乳动物细胞和植物的生化测定验证。编码候选相互作用的基因将通过高通量CRISPR-CAS9介导的反向遗传筛选进行研究，以识别KAI2依赖性信号活性的KUF1抑制器和调节剂。假定的KUF1靶标将测试植物中KUF1依赖性的多泛素化和降解。该奖项是由综合有机体系统划分的生理机制和生物力学计划共同资助的，以及分子和蜂窝生物科学部门的细胞动态和功能集群。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识优点和广泛的范围来评估的。

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.

Evolution of Plant Hormone Response Pathways

• DOI: 10.1146/annurev-arplant-050718-100309
• 发表时间: 2020-01-01
• 期刊: ANNUAL REVIEW OF PLANT BIOLOGY, VOL 71, 2020
• 作者: Blazquez, Miguel A.;Nelson, David C.;Weijers, Dolf
• 通讯作者: Weijers, Dolf

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

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.