In plants, communication between cells and organs occurs predominantly via small molecule signals, and this communication is required to integrate developmental and environmental signals into a coherent growth and reproductive strategy. More detailed understanding of small molecule signaling in plants will allow for rational improvements to crops and agricultural practices. A major limitation for understanding small molecule signaling is the lack of tools for measuring the fluctuations of small molecule concentrations in cells or subcellular compartments. Small signaling molecules (e.g. hormones) bind to specific receptor proteins, which then change shape (conformation). The altered receptor conformation leads to further signaling that eventually results in appropriate responses to the original hormone signal. In principle, the receptor protein conformation change induced by hormone binding can be used as a proxy for hormone concentration if the conformation change can be tracked. This tracking can be accomplished with high-resolution using Förster resonance energy transfer (FRET) nanosensors. FRET nanosensors are fluorescent proteins that change conformation upon small molecule binding. This conformational change results in FRET efficiency changes that can be measured optically and non-invasively. At least presently, the design of such sensors is empirical, requiring years of development and optimization for a single sensor without a guarantee of success. The central aim of this project is to rapidly develop a suite of new nanosensors for hormones using a microfluidic chip designed for high-throughput protein synthesis and fluorescence analysis. Thousands of potential hormone sensing proteins will be synthesized on microfluidic chips and then tested for FRET changes after exposure to plant hormones. Proteins that can track hormone concentration in vitro will be deployed in plants to verify that they can be used for dynamic hormone measurement in vivo. Hormone sensors are a critical and needed tool and the nanosensors developed in this project will be made available to the scientific community. The novel platform for rapid sensor development will be useful for constructing nanosensors for measuring many more small molecules, thereby dramatically expanding the potential tool sets available for studying signal perception and transduction. The project also provides a unique opportunity to cross-train a postdoctoral fellow in hormone biology and bioengineering. Finally, students from a local high school with a high minority representation will participate in the project and will test a simple low-cost digital camera-based FRET imaging system for non-invasive hormone analysis.
Plants are unique in that they are sessile and thus, over their individual lifespan (between months and thousands of years), have to successfully cope with the rapid changing and dynamic environment. Research in this area becomes more and more importance since climate change is leading to increased incidence and severity of extreme weather situations, including drought. Hormones play critical roles in communication of environmental conditions and control of plant acclimation. Massive progress has been made in the identification of hormone biosynthetic enzymes, transporters, and importantly also receptors. However, there is a critical piece of information missing: the distribution and dynamics of the plant hormones in live plants with cellular and subcellular resolution. This high-risk project built a platform for creating a tool – namely fluorescent sensors that can monitor cellular, and even intracellular dynamics of hormones in live plants with minimal invasion. The group successfully deployed this platform to create sensors for several plant hormones, which serves as a proof of concept that the sensor engineering pipeline can be expanded to all plant hormones. While this work was performed in plants, it has implications for the development of sensors for metazoan hormones and thus relevance in the context of medical research. The group created sensors for two of the important plant hormones, implemented them in plants and measured hormone distribution in intact plants. These sensors can now be used to carefully characterize hormone dynamics in live plants, to advance models of hormone action, test hypotheses regarding hormone distribution in response to environmental changes, 2. Broader impacts The work promises new insights into drought responses with much increased temporal and spatial resolution, and is expected to provide new insights into drought stress responses. Data generated with the new tool set are likely of fundamental importance for developing new approaches to make crop plants more tolerant to periods of drought. The project trained several students and postdoctoral scientists in technologies at the interface of physiology, protein engineering, cell biology, and biophysics.
