Unraveling a new signaling pathway for brassinosteroids: use of a novel biosensor for localized cAMP elevation to link cyclic nucleotide, Ca2+ and plant steroid signal transduction

类固醇激素调节动物和植物的生长发育。植物有一类类固醇激素，称为油菜素内酯（BR），除其他作用外，促进细胞伸长和细胞扩增。了解BR作为促生长激素的分子机制，可以为提高作物产量和品质提供新的途径。已知BR与植物细胞表面的受体结合后，诱导基因表达的变化，从而调节生长发育。除了这些基于基因表达变化的相对缓慢的BR反应外，研究小组最近还发现了由小的、可移动的分子（所谓的第二信使）引发的更快速的BR反应。该项目将研究BR如何与其受体结合导致细胞内第二信使浓度的增加，以及第二信使浓度的增加如何带来下游反应。为了实现这一目标，首席研究员已经开发并将进一步建立光学传感器，以高灵敏度记录活植物细胞内的第二信使浓度。因此，对BR如何影响植物细胞的基本新见解有望实现。第二种被研究的信使之一是cAMP（环腺苷单磷酸），它在动物细胞中作为信使已被确定，但在植物细胞中cAMP的合成和功能多年来一直是一个谜。本项目旨在为cAMP是植物细胞中真正的第二信使提供实验证据。该项目采用转基因（Glosensor）拟南芥植物，表达融合环状AMP （cAMP）结合域的萤火虫荧光素酶。这种生物传感器在cAMP结合时产生光，提供了一种独特的工具；这种植物可以实时监测胞质信号分子cAMP的局部升高。植物Ca2+传导离子通道被cAMP激活这些通道（以及激活它们的cAMP）在许多信号通路中发挥作用，包括最近发现的油菜素内酯（BR）信号通路。参与cAMP生成的植物蛋白与动物中的植物蛋白不同。编码这些酶的植物基因（以及相应的突变体）尚未被识别；Glosensor植物为研究基于camp的信号传导提供了另一种遗传工具。该项目将采用Glosensor植物和新开发的方法来监测膜定位的胞质Ca2+升高，以检查Ca2+依赖性BR信号的分子成分。信号级联被认为涉及G蛋白，以及钙调蛋白（CaM）和钙调蛋白依赖性转录激活因子（CAMTA）。这些蛋白在信号级联中的参与将使用适当的拟南芥突变体进行询问。一项创新的分子遗传学培训计划（Camp DNA）将提供给高中教师，作为更广泛的影响。这个为期两周的项目以综合的方式培训参与者的实验方法和基本原理。为参与者提供了一套工具和资源（信息和生物），以支持分子遗传学体验实验室高中教育计划。该奖项反映了美国国家科学基金会的法定使命，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。

Steroid hormones regulate growth and development in animals and plants. Plants have a class of steroid hormone called brassinosteroids (BR), which, among other effects, promote cell elongation and cell expansion. Understanding the molecular mechanisms, how BR acts as growth-promoting hormone, could provide new avenues to enhance crop yield and quality. It is known that upon binding to a receptor at the plant cell surface, BR induces changes in gene expression, which regulate growth and development. In addition to these relative slow BR responses based on changes in gene expression, more rapid BR responses triggered by small, mobile molecules, so-called second messengers, have recently been discovered by the research team. This project will investigate how BR binding to its receptor causes an increase in second messenger concentrations inside the cell, and how this increase in second messenger concentrations brings about downstream responses. To achieve this goal, the Principal Investigator has developed, and will further establish, optical sensors that allow recording second messenger concentrations with high sensitivity inside a living plant cell. As a result, fundamental new insights into how BR affects plant cells are expected. One of the second messengers to be studied is cAMP (cyclic adenosine monophosphate), which is well established as messenger in animal cells, but in plant cells synthesis and function of cAMP have been an enigma since many years. This project is aimed at providing experimental evidences that cAMP is a bona fide second messenger in plant cells.The project employs transgenic (Glosensor) Arabidopsis plants that express firefly luciferase fused to a cyclic AMP (cAMP) binding domain. This biosensor generates light upon cAMP binding, providing a unique tool; plants that allow real-time in vivo monitoring of localized elevations of the cytosolic signaling molecule cAMP. Plant Ca2+ -conducting ion channels are activated by cAMP; these channels (and the cAMP that activates them) play roles in numerous signaling pathways, including a newly uncovered role in brassinosteroid (BR) signaling. Plant proteins involved in cAMP generation are not canonical to those in animals. Plant genes encoding these enzymes (and corresponding mutants) are not identified; Glosensor plants provide an alternative genetic tool to study cAMP-based signaling. The project will employ Glosensor plants, and newly developed methods to monitor membrane-localized cytosolic Ca2+ elevations to examine molecular components of Ca2+ -dependent BR signaling. The signaling cascade is thought to involve G proteins, as well as calmodulin (CaM) and a CaM-dependent transcriptional activator (CAMTA). Involvement of these proteins in the signaling cascade will be interrogated using appropriate Arabidopsis mutants. An innovative molecular genetics training program (Camp DNA) will be delivered to high school teachers as a broader impact. This two-week program trains participants in experimental methods and underlying principles in an integrated fashion. Participants are provided with a package of tools and resources (informational and biological) to support a molecular genetics experiential lab high school educational program.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.