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

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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.