1. Cell surface carbonate chemistry.
We have completed a detailed experimental microelectrode monitoring and modelling analysis of total carbonate chemistry at the cell surface of a photosynthetic diatom. This work has revealed dramatic fluctuations in pH and carbonate at the surface of a large diatom as a direct response to photosynthetic carbon uptake. In contrast, small diatoms that do not suffer from diffusion limitation at their cell surface do not show such pronounced fluctuations. We have shown that for large cells the changes in chemistry are far more rapid and pronounced at the cell surface than in the bulk medium, forcing a reconsideration of phytoplankton productivity models that rely on bulk seawater parameters.
2. Molecular tools development.
We have generated a range of genetically encoded fluorescent reporters expressed in diatoms. These include reporters for cytosolic calcium, chloroplast calcium cellular pH, cellular phosphate, membrane potential and reactive oxygen generation in cytosol and chloroplast. These are now being successfully applied in many aspects of the project. New findings have shown a key role for plasma membrane NADPH oxidase enzymes in the generation of extracellular reactive oxygen species and the alleviation of oxidative stress arising from photosynthesis in diatoms.
We have also established gene knock out approaches for ion channels in diatoms, allowing for the first time detailed functional analysis of their functions.
3. Calcium signalling.
We have carried out extensive analysis of calcium signalling in response to a range of external cues in the diatom P. tricornutum. This work is providing new insights into the regulation of cytosolic calcium and its role in signalling in in response to osmotic stress, light temperature changes and nutrient limitation.
4. A new class of eukaryote ion channels.
A major finding is the discovery of a novel class of eukaryotic voltage-dependent cation channels (VDCCs) in diatoms and coccolithophores. These have close similarity to primitive bacterial voltage-dependent sodium channels (BacNav). We have shown that these single pore domain channels serve to impart electrical excitability to diatoms in a manner similar to the way in which the more complex 4-domain voltage-dependent sodium or calcium channels operate in animal cells,providing an alternative mechanism for fast electrical excitability.. By using electrophysiological characterization in a heterologous expression system (human HEK cells) we have shown unexpectedly that the EukaCat channels from the diatom P. tricornutum (EukCatA) behave as calcium channels whereas the coccolithophore EukCat channels (EukCatB) behaves as sodium channels. These results are allowing new insights into the evolution of channel selectivity in eukaryotes. They indicate that the development of 4-domain Navs and Cavs was not a pre-requisite for the evolution of sophisticated rapid signalling processes in eukaryotes. These novel classes of ion channels represent excellent new tractable models for the future study of ion channel structure and function.
5. Studies of natural populations.
We have participated in a major oceanographic cruise to the Great Calcite Belt of the Southern Ocean. This provided the unique opportunity to apply methods for single cell analysis of phytoplankton cells to larger natural populations. The cruise has been a success and a large amount of novel cell physiological data, including full characterisation of photosynthetic efficiency parameters of single cells an population samples has been obtained.
