EAGER:Novel approaches to study green leaf volatile perception

被食草动物伤害的植物会释放挥发性有机化合物(VOC)到周围的植物可以感知到的空域中。这些植物将挥发性化合物解释为警报信号，并开始上调对敌人的防御反应，为即将到来的攻击做好准备。挥发性化合物作为防御诱导剂的作用已经有了很好的描述，但植物如何感知它们还不清楚。一般来说，警报信号是由特定的受体蛋白感知的。大多数食草动物诱导的挥发性化合物都很小，很难追踪，这阻碍了对其受体的识别。该项目将开发一种新的方法，用非常小的标签标记挥发性化合物，可以用来跟踪化合物，从而识别它们的受体。标记策略可以适用于其他小型信息化学品，从而改变我们对植物交流的知识。识别挥发性警报信号的受体蛋白可能有助于通过依赖天然植物防御而不是农用化学品来设计可持续作物生产的新策略。这个项目的各个方面也将被纳入到生态学与进化实验室课程中。此外，将积极招收STEM学科中代表性不足的本科生，提供课外体验式学习机会。这些学生将研究与本项目相关的VOC信令。这将培养他们的实践、分析和沟通技能，为他们的职业生涯做好准备。此外，两名研究生将接受生物学和化学方面的培训，以提供化学生态学和分子生物化学方面的前沿交叉学科教育。植物交流的许多方面是由绿叶挥发物(GLV)等挥发性有机化合物介导的。当植物受到食草动物的伤害时，它们会释放出GLV，这不仅可以被受伤植物的远端部分感知(植物内部的通讯)，也可以被邻近的植物感知(植物间的通讯)。受体组织对这些GLV的感知被解释为迫在眉睫的危险的迹象，并导致防御反应的启动或上调。这项急切的提议的目标是推进新的方法来识别启动防御信号的GLV受体蛋白。像GLV这样的非常小的非蛋白质配体(只包含六到八个碳)很难标记和跟踪。这项建议采用了一种新的方法，使用“点击化学”将具有生物活性的微型GLV连接到报告标签上，以识别GLV的受体蛋白。发出荧光的记者将被用于定位受体-GLV-报告分子复合体，基于生物素的报告分子标记将被用于纯化该复合体并分离GLV受体。此外，GLV诱导的基于快速磷酸化的信号事件将通过磷酸蛋白质组学方法确定，以识别早期GLV响应信号蛋白，如GLV受体。尽管GLV在植物信息交流中的作用已经得到了很好的证明，但植物如何感知GLV仍然是完全未知的。这是一个重大的知识差距，阻碍了我们对GLV信号和功能的理解。GLV受体的识别和表征将在分子和生态水平上改变我们对植物通讯的理解。这一奖项反映了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。

Plants that are injured by herbivores emit volatile organic compounds (VOC) into the airspace which can be perceived by neighboring plants. These plants interpret the volatile compounds as alarm signals and start to upregulate defense responses against their enemies to be prepared for an impending attack. Effects of volatile compounds as inducers of defenses are well characterized, but it is unknown how plants perceive them. In general, alarm signals are perceived by dedicated receptor proteins. Most herbivore-induced volatile compounds are very small and difficult to track, which impedes the identification of their receptors. This project will develop a novel approach for labeling volatile compounds with very small tags that can be used to track the compounds and thus identify their receptors. The tagging strategy can be adapted for other small infochemicals and thus transform our knowledge of communication in plants. Identification of receptor proteins for volatile alarm signals may help to devise novel strategies for sustainable crop production by relying on innate plant defenses rather than agrochemicals. Aspects of this project will also be incorporated into an Ecology and Evolution laboratory class. In addition, undergraduates from groups underrepresented in STEM disciplines will be actively recruited to provide extra-curricular experiential learning opportunities. These students will work on VOC signaling related to this project. This will develop their practical, analytical, and communication skills, which prepares them for professional careers. In addition, two graduate students will be trained in biology and chemistry to provide a cutting-edge interdisciplinary education in chemical ecology and molecular biochemistry.Many aspects of plant communication are mediated by volatile organic compounds such as green leaf volatiles (GLVs). When plants are injured by herbivores, they emit GLVs, which can be perceived not only by distant parts of the wounded plants (within-plant communication) but also by neighboring plants (interplant communication). Perception of these GLVs is interpreted by receiver tissues as a sign of imminent danger and results in priming or upregulation of defense responses. The objective of this EAGER proposal is to advance novel approaches to identify GLV receptor proteins that initiate defense signaling. Very small non-proteinaceous ligands like GLVs (containing only six or eight carbons) are difficult to tag and to track. This proposal takes a novel approach by using ‘click chemistry’ to couple bioactive minitagged GLVs to reporter tags in order to identify receptor proteins for GLVs. Reporters that emit fluorescent light will be used to localize the receptor-GLV-reporter complex, and biotin-based reporter tags will be used to purify this complex and to isolate a GLV receptor. Additionally, GLV-induced rapid phosphorylation-based signaling events will be determined by a phosphoproteomics approach to identify early GLV-responsive signaling proteins such as GLV receptors. Although the role of GLVs in plant communication is well documented, it is completely unknown how they are perceived by plants. This is a significant knowledge gap that hampers our understanding of GLV signaling and function. Identification and characterization of GLV receptors would transform our understanding of plant communication at the molecular and ecological level.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.