Small molecules such as calcium ions (Ca2+) or reactive oxygen species (ROS) play an essential role in the early signaling events that relay information on environmental changes into cellular responses. In complex signaling networks these so-called second messengers regulate and interact with other signaling pathways, e.g. phytohormones. To better understand how these early signaling events affect the acclimation to stress conditions in potato, systems to measure dynamic real-time changes in intracellular calcium concentrations, ROS and several plant hormones directly in potato crops were developed in a collaboration of partners from the University of Bonn, the FAU Erlangen, Durham University, the National Institute of Biology in Ljubljana and Solana Research GmbH.
Two in vivo measurement systems were established: i) sensor lines that directly measure changes in free Ca2+ and ROS and ii) gene reporter lines, which use promoter-reporter fusion constructs to indirectly measure signaling events via their down-stream gene induction.
- Responses of the sensors and luciferase reporters are measured using camera (Durham Unviersity) or plate reader-based techniques (University of Bonn). Fluorescence-based reporters but also roGFP are analysed by confocal laser scanning microscopy (Durham, Ljubljana, Bonn).
- The response of the Ca2+ sensor line to different stimuli such as H2O2, salt, osmotic stress, or flg22 have been analysed successfully using plant grown in laboratory growth chambers. A reporter line based on the StRD29 promotor has been used for a case study on the in vivo effects of combinatorial hormone applications.
- The suitability of various gene reporter lines for integration into HTP screening platforms is currently being tested by PSI.
These sensor and reporter lines will become an integral part of our extensive studies on early signaling events in the chosen potato variety Désirée under drought/heat and flooding stress to ultimately generate a detailed timeline and comprehensive event map of early stress responses.
