EAGER: Development of Fluorescence-based Nanosensors for Dynamic Measurement of Active Cytokinins in Plants

PI: G. Eric Schaller (Dartmouth College)

CoPIs: Joseph J. Kieber (University of North Carolina-Chapel Hill) and Sakiko Okumoto (Virginia Polytechnic Institute and State University)

Cytokinins are plant hormones that regulate diverse growth and development processes, such as cell division, metabolism, chloroplast development and maintenance, and senescence, as well as responses to biotic and abiotic factors. In spite of their critical role in plants, no method has yet been developed to accurately determine the cellular and subcellular dynamics of cytokinin levels. This EAGER project seeks to develop fluorescence-based nanosensors capable of measuring cytokinin levels in a monocot (rice) and a dicot (Arabidopsis). FRET- and single fluorescent protein-based nanosensors will be developed and tested using the cytokinin-binding CHASE domain from the Arabidopsis cytokinin receptor AHK4 and the corresponding domain of the rice OsHK4 receptor as ligand-binding domains. The requirements for cytokinin binding by the CHASE domain using a radioactive cytokinin-binding assay will be assessed to facilitate nanosensor design. Nanosensors will initially be tested in vitro, making use of bacterially expressed fusion proteins and subsequently be tested in planta, initially using a transient protoplast transformation system and then by generation of stable transgenic lines. The response to exogenous cytokinin and to altered levels of endogenous cytokinin will be tested to confirm the ability of the nanosensors to report cytokinin levels in vivo. Finally, using different targeting sequences, nanosensors will be targeted to the cytosol and the endoplasmic reticulum to explore how cytokinin levels vary within the cell and the dynamics of cytokinin movement in response to various inputs. This is of particularly importance for cytokinin because its receptors are localized to the ER membrane, and the site of perception is within the ER lumen. If successful, this project will allow for the quantification of bioactive cytokinins at the cellular and subcellular levels and for dynamic measurements in living cells.

Cytokinins play key roles in regulating many plant properties of agricultural significance. For example, cytokinins play a critical role in regulating grain yield in rice. Development of nanosensor technology will facilitate and advance studies to improve these cytokinin-regulated traits. Vectors and transgenic Arabidopsis and rice lines for the nanosensors will be made available to the research community on request and/or through appropriate stock centers including the Arabidopsis Biological Resource Center. The project will enhance the infrastructure of research and education by providing hands-on interdisciplinary training for undergraduate and graduate students as well as postdoctoral research associates.