1 Department of Systems Biology, Technical University of Denmark2 Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark3 Department of Micro- and Nanotechnology, Technical University of Denmark4 Bioanalytics, Department of Micro- and Nanotechnology, Technical University of Denmark5 Fluidic Array Systems and Technology, Department of Micro- and Nanotechnology, Technical University of Denmark6 Proteomics Platform, Department of Systems Biology, Technical University of Denmark7 Center for Intelligent Drug Delivery and Sensing Using Microcontainers and Nanomechanics, Center, Technical University of Denmark
This project focuses on developing and applying a tissue culture system with electrochemical and optical detection techniques for tissue culture of barley aleurone layer to increase understanding of the underlying mechanisms of programmed cell death (PCD) in plants. The major advantage of electrochemical detection systems is that they can be miniaturized, multiplexed and automated without losing their performance[1,2]. Combining tissue culture with electrochemical and optical detection allows implementation of a wide range of assays for online, real-time, parallel analysis of important parameters such as redox activity, O2 and H2O2 concentration, pH, cell viability and release of target enzymes such as α-amylase. We have optimised an intracellular, whole-cell redox activity assay that detects changes in redox activity in barley aleurone layer during PCD. The assay uses a double mediator-system to electrochemically measure redox activity via changes in the NADP:NADPH ratio. Experiments show that redox activity changes depend on phytohormone activation or inactivation of aleurone layer metabolism and subsequent PCD. We have also successfully detected PCD induced by phytohormones in barley aleurone layer using an optical double-fluorescent probe-system. Currently, we are working on integrating both detection methods into a tissue culture system for immobilised plant tissues.