TRTech-PGR: Mass Spectrometry Imaging of in vivo Isotope labeling

在分子水平上了解植物对病原体或不利生长条件的反应对于作物生物工程至关重要，因为全球化和气候变化带来的挑战越来越大。 该项目开发的新生物技术工具将使植物生物学家能够在时间和空间维度上可视化细胞水平的代谢变化。该项目的重点是开发工具，以了解单细胞分辨率的动态植物代谢;然而，潜在的影响超出了植物生物学的许多科学学科，包括微生物生物技术，精密医学和环境应用。除了在生物科学和物理科学之间的跨学科领域培训研究生和本科生外，每年还将通过爱荷华州州立大学的教师研究经验计划招募一名中学或高中教师参加该项目。 教师们将参与一个与环境有关的浮萍项目，他们可以在回到教室后继续开展自己的项目。将开发的技术是体内同位素标记的质谱成像（MSI），简称为MSIi。 体内同位素标记可以追踪代谢活动，并提供代谢变化的时间信息。同位素标记的代谢物分布然后可以通过MSI以细胞分辨率直接在组织上可视化。水培文化将优化体内同位素标记的三种模式植物，浮萍，拟南芥，和玉米。 用2 H、15 N或13 C标记的植物组织的MSI数据将用于直接鉴定组织上的未知代谢物，并可视化选定生物系统中的代谢物通量。 为了以自动和稳健的方式加快数据分析，将通过在现有的开源软件MSiReader中添加多种功能来开发软件工具，例如校正同位素测量误差，生成同位素图像和丰度，可视化标记效率，以及实现非目标标记搜索。 一旦成功，新技术可以扩展到许多新的方向，包括单细胞分辨率的通量组学，高精度代谢生物工程和细胞或组织特异性动态应力响应。该奖项反映了NSF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。

Understanding plant metabolism at the molecular level in response to pathogens or hostile growth conditions is crucial for the bioengineering of crop plants with the growing challenges imposed by globalization and climate change. The new biotechnological tools to be developed in this project will allow plant biologists to visualize cellular level metabolic changes in both temporal and spatial dimensions. This project is focused on developing tools to understand dynamic plant metabolism at single-cell resolution; however, the potential impact extends beyond plant biology across many scientific disciplines, including microbial biotechnology, precision medicine, and environmental applications. In addition to training graduate and undergraduate students in this interdisciplinary field between biological and physical sciences, a middle or high school teacher will be recruited to this project each year through the Research Experiences for Teachers program at Iowa State University. Teachers will work on an environment-related duckweed project, which they can continue after going back to the classroom and develop their own projects.The technology which will be developed is mass spectrometry imaging (MSI) of in vivo isotope labeling, referred to as MSIi. In vivo isotope labeling can trace metabolic activities and provide temporal information of metabolic changes. Isotope labeled metabolite distributions can then be visualized by MSI directly on tissues at cellular resolution. Hydroponic cultures will be optimized for in vivo isotope labeling of three model plants, duckweed, Arabidopsis, and maize. MSI data of plant tissues labeled with 2H, 15N, or 13C will be used to identify unknown metabolites directly on tissues and visualize metabolite flux in selected biological systems. To expedite the data analysis in an automatic and robust fashion, software tools will be developed by adding multiple functionalities to the existing open-source software, MSiReader, such as correcting isotope measurement errors, generating isotopologue images and abundances, visualizing labeling efficiency, and enabling untargeted labeling search. Once successful, the new technology can be expanded to many new directions, including fluxomics at single-cell resolution, high-precision metabolic bioengineering, and cell- or tissue-specific dynamic stress responses.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.