Process Understanding
MIMS bioassays provide detailed information on processes like net/gross photosynthesis, CO2/HCO3- uptake and efflux, intra/extracellular CA activities, respiration including chlororespiraton (CR), mehler reaction (MR), photorespiration (PR), dark respiration (DR), electron transport rate and energy capture efficiency.
To develop a process-based understanding of the observed responses, we apply different in vivo bioassays. Membrane-inlet mass spectrometry (MIMS) allows monitoring of gas exchange processes of phytoplankton on a cellular level in real-time. Our MIMS system consists of a custom-made cuvette and inlet system combined with a sectorfield multicollector mass spectrometer. Dissolved gas molecules like CO2 or O2 permeate through the membrane and are ionised and detected only seconds later in the mass spectrometer. The advantage of this approach is that several processes can be observed and quantified simultaneously.
A suite of methods allows quantification of cellular C fluxes, a prerequisite to understand the effect of CO2 on photosynthesis, growth and other down-stream processes. One method allows to distinguish between CO2 and HCO3- as carbon sources and determines the uptake kinetics as a function of C availability or other environmental conditions. In another application, the use of stable isotopes allows to determine activities of carbon anhydrase, a key enzyme catalyzing the otherwise slow interconversion between CO2 and HCO3-.
In addition to C fluxes it is essential to also unravel the energy fluxes of the cell. Using different stable isotopes as tracers, oxygen consuming and evolving processes during photosynthesis can be separated. In addition to estimates of gross photosynthesis (photosynthetic electron transport) and thus energy entering the system, this approach also provides information on light-dependent processes like the Mehler reaction, chlororespiration or photorespiration. These processes can strongly modulate light and CO2 dependence in photosynthesis by altering the flow of electrons from PSII to carbon fixation.
To go beyond this level of information we combined MIMS measurements with fast-repetition rate fluorometry using a fluorescence-induction relaxation system (FIRe). The FIRe provides a unique analytical insight into photosynthesis by employing both single turnover and multiple turnover flashes. As examples, the FIRe technique derives the functional absorption cross section, the extent of excitation transfer between PSII reactions centres (connectivity factor), the yield of charge separation (efficiency of energy capture), and kinetics of photosynthetic electron transport. The combined assessment of a bio-optical proxy of electron transport with a direct measure of carbon fixation and O2 evolution provides a detailed understanding of the processes which cause sensitivity to environmental conditions affected by global change.
Related publications: Kranz et al. 2011, Kranz et al 2010, Levitan et al. 2010, Engel et al. 2010, La Roche et al. 2010, Kranz et al. 2009, Rost et al. 2007, Rost et al. 2006, Rost et al. 2003.
