Integrating the Regulatory Components of Sphingolipid Biosynthesis in Arabidopsis

鞘脂作为质膜、张力体和膜内系统的主要结构成分，有助于蛋白质运输和膜的组织和物理性质。鞘脂还作为基本细胞过程的动态调节剂，包括程序性细胞死亡（PCD），这是由生物活性鞘脂代谢物长链碱基和神经酰胺的积累引发的。该项目旨在阐明调节成分及其相互作用，这些成分精细地调节足量鞘脂的需求，以支持细胞活力和生长，同时防止鞘脂代谢物积聚到诱导PCD的水平。该项目将建立在通过丝氨酸棕榈酰转移酶（SPT）对鞘脂合成的翻译后调节的新证据基础上，SPT是一种异二聚体酶，催化了该途径的第一步。拟南芥在SPT调控网络中的新参与者的同源物将被表征。其中包括刺激SPT活性的SPT小亚基（ssSPT）和响应细胞内鞘脂水平对SPT活性提供可逆负调控的orm。拟南芥ssSPT和ORM基因家族成员对SPT和鞘脂合成调控的贡献将被确定。利用基于酵母的工具，将评估拟南芥orm的翻译后修饰，并确定SPT和调节蛋白的相互作用。还将进行实验，以确定SPT监管体系的任何其他组成部分。基于酵母的研究结果将为揭示SPT在植物体内对鞘脂稳态扰动的调控转导提供实验基础。这些研究将利用鞘脂生物合成调节功能障碍的拟南芥突变体和真菌毒素，这些真菌毒素会产生高水平的诱导pcd的长链碱基。阐明控制鞘脂合成的机制不仅对作物产量有影响，而且由于鞘脂在所有真核细胞中都是必需的和高度生物活性的，因此具有广泛的普遍意义。更广泛的影响：该项目包括研究生和本科生培训以及高中教育推广。这项研究将由内布拉斯加大学林肯分校（UNL）和卫生科学统一服务大学（USUHS）的研究生进行。每年两至四周的研究生交换机会将为参与的学生拓宽培训经验。为了满足UNL不断增长的教育需求，在PI Cahoon和co-PI Stone的领导下，将建立一个研究生水平的脂质生物化学团队授课课程。本课程将包括鞘脂功能和代谢模块，并将广泛吸引对营养学，生物燃料和脂质信号感兴趣的学生。本科培训将通过UNL本科生创造性活动和研究经验（UCARE）计划纳入该项目，该计划为本科生提供为期两年的开发和开展独立研究项目的机会。与项目目标一致的本科培训也将通过在UNL建立的NSF REU项目和USUHS的科学和技术指导项目提供，这些项目积极招收女性和代表性不足的少数民族学生。该项目还将通过一年一度的UNL女性科学项目的实验室研讨会，以及内布拉斯加州epscor支持的内布拉斯加州青年科学家计划（一个旨在加强K-12 STEM学生管道的暑期项目），为高中生提供教育推广服务。

Sphingolipids contribute to protein trafficking and the organization and physical properties of membranes as major structural components of the plasma membrane, tonoplast, and endomembrane system. Sphingolipids also act as dynamic regulators of basic cellular processes, including programmed cell death (PCD), which is triggered by the accumulation of the bioactive sphingolipid metabolites long-chain bases and ceramides. The project is designed to elucidate the regulatory components and their interactions that finely mediate the requirements for sufficient amounts of sphingolipids to support cell viability and growth yet prevent the buildup of sphingolipid metabolites to levels that induce PCD. The project will build upon emerging evidence for post-translational regulation of sphingolipid synthesis through serine palmitoyltransferase (SPT), a heterodimeric enzyme that catalyzes the first step in this pathway. Arabidopsis homologs of new players in the SPT regulatory network will be characterized. These include small subunits of SPT (ssSPT) that stimulate SPT activity and ORMs that provide reversible negative-regulation of SPT activity in response to intracellular sphingolipid levels. Contributions of members of Arabidopsis ssSPT and ORM gene families to the regulation of SPT and sphingolipid synthesis will be determined. Using yeast-based tools, post-translational modifications of Arabidopsis ORMs will be assessed and interactions of SPT and regulatory proteins will be determined. Experiments will also be conducted to identify any additional components of the SPT regulatory system. Results from the yeast-based studies will provide the basis for experiments designed to uncover the in planta transduction of SPT regulation in response to perturbations in sphingolipid homeostasis. These studies will take advantage of Arabidopsis mutants with dysfunctions in sphingolipid biosynthetic regulation and fungal mycotoxins that generate enhanced levels of PCD-inducing long-chain bases. Elucidation of the mechanisms responsible for controlling the synthesis of sphingolipids will not only have implications for crop productivity but will also be of broad general interest since sphingolipids are both essential and highly bioactive in all eukaryotic cells.Broader Impacts:The project encompasses graduate and undergraduate training and high school educational outreach. The research will be conducted by graduate students at University of Nebraska-Lincoln (UNL) and the Uniformed Services University of the Health Sciences (USUHS). Opportunities for an annual two-to-four week exchange of graduate students between UNL and USUHS will broaden training experiences for student participants. To meet a growing educational need at UNL, a graduate level, team-taught course in lipid biochemistry will be established under the leadership of PI Cahoon and co-PI Stone. This course will include modules on sphingolipid function and metabolism and will broadly engage students with interests ranging from nutrition, biofuels, and lipid signaling. Undergraduate training will be incorporated into the project through the UNL Undergraduate Creative Activities and Research Experiences (UCARE) program that provides a two year opportunity for undergraduate students to develop and conduct independent research projects. Undergraduate training in line with the project goals will also be provided through established NSF REU programs at UNL and science and technology mentorship programs at USUHS, which actively recruit women and underrepresented minority students. The project will also facilitate educational outreach to high school students through lab-based workshops for the annual UNL Women in Science program and by hosting of high school students in the Nebraska EPSCoR-supported Young Nebraska Scientist Initiative, a summer program designed to strengthen the K-12 STEM student pipeline.