H2O2 is a reactive oxygen species that occurs naturally as a by-product of photosynthesis. This poses a problem for photosynthetic cells: when H2O2 accumulates, it reacts randomly with substances in the immediate environment and can damage both the proteins that keep photosynthesis going and the DNA of the chloroplast. At the same time, however, H2O2 may also be an important messenger substance. Compared to other reactive oxygen species, H2O2 is relatively stable, which makes the possibility of exporting it into the cytoplasm and thus using it as a signaling substance conceivable. Signals that the chloroplast sends back to the nucleus are called ‘retrograde’ - such retrograde signaling of how the chloroplast is doing is very important, because the vast majority of the chloroplast's proteins are encoded in the nucleus, produced in the cytoplasm and then imported into the organelle. If the chloroplast requires a different composition of proteins, for example because considerably more light suddenly arrives, the nucleus must be informed in order to create a balance.
In order to test the activity of H2O2 in the cell, biosensors have been developed in recent years on the basis of fluorescent proteins, which are able to indicate how much H2O2 is present via a fluorescence signal. José Ugalde's group at INRES has upgraded its fluorescence plate readers with a module that enables the samples to be irradiated with light in the machine for the application of such biosensors in photosynthesis research. This makes it possible to directly measure how much H2O2 is produced when the cells are exposed to a certain amount of light. By creating different strains, each with the biosensor in different cell compartments, it is then possible to follow in real time how H2O2 is produced in the chloroplast through photosynthesis and then spreads to other parts of the cell.
In order to make optimal use of these technologies, we in RG Caspari aim to clone new gene constructs that generate the H2O2 sensor ‘Hyper7’ in different cell compartments of the green alga Chlamydomonas reinhardtii. We can then compare these strains with existing H2O2 sensor strains in algae and plants. We also want to use the biosensors to investigate different mutants that lack photosynthesis or H2O2 degradation genes. With this work, we want to better determine the role of H2O2 as a retrograde signal, consolidate the cooperation between the two laboratories and collect data in order to write a publication and obtain further funding.