Objective
The objective of this project is to understand how photoacclimation is regulated in dynamic environments, leading to unbalanced growth. Technique development, application of techniques to monitor primary production in the Oosterschelde and Westerschelde estuary and the molecular biology related to photoinhibition is part of this project.
Approach
Continuous cultures, limited by either light or nutrients and with dynamic light fields simulating vertical mixing, are studied and the photobiological characteristics are measured, together will cell density, and macromolecular composition. Photosynthesis is studied by simultaneous measurements of oxygen exchange using Membrane Inlet Mass Spectrometry (MIMS), 14C-uptake and variable chlorophyll fluorescence. Special emphasis will be placed on quantifying the relationship between the minimal fluorescence (Fo and Fo') and the functional cross section of PSII and at the existence of alternative electron sinks at high irradiance. Another important process is the occurrence of C-cycling. If bicarbonate is taken up by a carbon concentrating mechanism in excess of the demand for C-fixation, the CO2 formed by the carbonic anhydrase might leak out of the cells. The importance of this process will be measured (MIMS and IRMS) because it has important implications for fractionation patterns in algae (d13C). There is also evidence that C-cycling is fuelled by PSII driven electron transport, and that this can be an important aspect in the non-linear behaviour of rETR and C-fixation at high light. The developed techniques will also be used to study primary productivity in the field.
Results
MIMS measurements using 18O2 as a tracer for oxygen uptake in the light demonstrated that all eukaryotic algae studied showed light stimulated respiration, and that the stimulation takes place in the light limited region of a photosynthesis light curve. Stimulation of oxygen uptake in the light is generally 2-3-fold compared to the respiration in the dark. This stimulated oxygen uptake is not only due to increased mitochondrial respiratory electron transport, but is also due to the Mehler reaction (donation of electrons to O2 by PSI). The stimulated oxygen uptake was rather insensitive to the external DIC concentration, which is evidence that the light stimulated oxygen uptake is mainly due to the Mehler reaction, and that photorespiration plays a limited role. The effect of light stimulated respiration on total daily primary productivity is limited: it decreased the net productivity by 4-8% in comparison to a situation with a constant rate of oxygen uptake. That the effect is limited is mainly due to the fact that respiration in the dark part of water column and during the night is much larger.
Planning 2003
Estimation of the contribution of oxygen consuming reactions in the light, using the MIMS technique, will be carried out be doing measurements on both cultures and samples from field sites.
Special emphasis will be placed on the separation of the Mehler reaction and oxygen consumption due to photorespiration. Because of the high rates of Mehler reaction observed with dilute batch cultures, experiments will now be done using light and nutrient limited continuous cultures. Quantification of ETR will studied by developing methods to measure the optical (a*PSII) and functional PSII cross section (sPSII), and to investigate the relationship between Fo, a*PSII, sPSII and changes in the PSI cross-section using PSI absorption changes. These cultures will also be used to study how long the maximum quantum efficiency (Fv/Fm) will remain low in steady state nutrient limited cultures, as there is evidence in the literature that after many generations Fv/Fm will restore itself. New developments to be started will be variable fluorescence measurements on single cells and single cell absorption. To test the nature of the growth-limiting nutrient, nutrient induced fluorescent transients (NIFTs) will be carried out on whole samples and on single cells. In 2003 we will also start with studying the molecular biology of photoinhibition. We have obtained evidence that benthic diatoms are more resistant to photoinhibition than pelagic diatoms. We will start with sequencing the psbA-gene of the diatoms present in our culture collection. The sequence information is needed to follow the presence during a tidal cycle of different species using psbA-sequences and to correlate the resistance to photoinhibition with vertical migration patterns of different species. In cooperation with RIKZ the Fast Repetition Rate Fluorometry (FRRF) technique will be studied as an alternative method to measure primary productivity and comparisons will be made with the 14C-technique and the PAM-technique, both on cultures and phytoplankton communities.
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