An ion-based pacemaker at the pollen tube tip: mechanism and functions in apical growth, guidance and cell-cell communication

花粉管尖端的离子基起搏器：在顶端生长、引导和细胞间通讯中的机制和功能该项目将研究花粉管中产生的离子和电信号的作用，花粉管是一种高度专业化的细胞，参与开花植物的繁殖。这些细胞是顶端生长细胞的代表，除了花粉管，还包括哺乳动物神经系统的真菌菌丝和发育中的神经元。具体来说，这些离子信号是协调花粉管生长和受精所需功能所必需的。这些结果不仅有助于预测和操纵导致种子生产的细胞行为，具有潜在的农业影响，而且还有助于发现电信号可以定义细胞形状，引导和扩展到植物细胞之外的通信的新原理。这项研究将涉及不同STEM领域的发展和整合，包括细胞生物学、遗传学、计算方法和数学建模。从该研究中开发的数据分析管道和新算法将在开源统计编程语言R中公开提供，允许分析复杂的振荡和细胞生长成像。教育和社会影响包括两名博士后的专业发展，本科生的培训，以及对当地高中生的指导。此外，一些外联和教育活动是该项目的预期成果，在这些活动中，吸引更广泛兴趣的结果和原始方法将向各种各样的受众介绍。高等植物的受精需要雄性配子体（花粉管）生长到雌性组织中，并找到可用的胚珠，最后破裂以传递精细胞。花粉管是高度极化的细胞，在某些物种中具有自然界中已知的最快的细胞生长速度。这种强劲的增长是由特定的空间和时间编排维持的，通常涉及集中在尖端的细胞内离子浓度的稳定振荡，而这又由细胞外离子通量产生。本项目要测试的主要假设是，存在一个离子基中央起搏器，负责花粉管中分钟范围内的振荡，其(1)由通道活性和膜电位之间的负反馈回路产生，(2)在细胞水平上协调宏观过程（例如细胞极性，根尖生长，引导），(3)参与生长花粉管之间的细胞间竞争/合作；(4)影响与雌性配子体的细胞间通讯。第一个目标是使用定制的电生理学工具和基因编码探针（Ca2+, H+， Cl-和膜电位）的实时成像来表征所有参数的振荡。这些将允许比较不同物种/生态型的特征以及编码拟南芥通道和转运体的基因家族的单个和多个突变体。振荡的特征将由为该项目开发的数据分析工具确定，从而可以建立数学模型。具体的预测将从微分方程模型中得到不同程度的细节，而实验验证将通过突变、药理学和体内受精方案等测试预测来实现。

An ion-based pacemaker at the pollen tube tip: mechanism and functions in apical growth, guidance and cell-cell communicationThe project will study the role of ion and electric signals generated within the pollen tube, a highly specialized cell involved in the reproduction of flowering plants. These cells are representative of apical growing cells, a group that in addition to pollen tubes, includes fungal hyphae and developing neurons of the mammalian nervous system. Specifically these ion signals are needed to coordinate pollen tube growth and function required for fertilization. The results will not only contribute to predicting and manipulating cellular behavior leading to seed production, with potential agricultural impacts, but also to discovering new principles by which electric signals can define cell shape, guidance and communication extending beyond plant cells. This investigation will entail the development and integration of different STEM areas, including cell biology, genetics, computational methods and mathematical modeling. A data analysis pipeline and novel algorithms developed from the research will be made publicly available in R, an open-source statistical programming language, allowing analysis of complex oscillations and cell growth imaging. Educational and societal impacts include professional development of two postdoctoral researchers, undergraduate student training, and mentoring of local high school students. In addition, a number of outreach and educational activities are expected outcomes of the project, where the results and original approaches appealing to broader interests will be presented to a wide variety of audiences.Fertilization in higher plants requires the male gametophyte (pollen tubes) to grow into the female tissues and locate available ovules, finally bursting to deliver the sperm cells. Pollen tubes are highly polarized cells and in some species have the fastest cellular growth rates known in nature. This vigorous growth is sustained by specific spatial and temporal choreographies, often involving stable oscillations, of intracellular ion concentrations focused at the tip, which in turn are generated by extracellular ion fluxes. The main hypothesis to be tested in this project poses the existence of an ion-based central pacemaker responsible for minute-range oscillations in pollen tubes, which (1) are generated by a negative feedback loop between channel activity and membrane potential, (2) coordinate macroscopic processes at the cellular level (e.g. cell polarity, apical growth, guidance), (3) are involved in cell-cell competition/cooperation between growing pollen tubes and (4) impact cell-cell communication with the female gametophyte. The first goal is to characterize oscillations in all parameters using custom-made electrophysiology tools and live imaging with genetically encoded probes (Ca2+, H+, Cl- and membrane potential). These will allow comparison between signatures of different species/ ecotypes and single and multiple mutants of gene families coding for channels and transporters in Arabidopsis. The features of the oscillations will be determined with data-analysis tools developed for the project, allowing the formulation of mathematical models. Specific predictions will then be derived from differential equation models in varying levels of detail, whereas experimental validation will be achieved by testing the predictions with mutations, pharmacology and in vivo fertilization protocols, among others.