Periodic Reporting for period 4 - SEACELLS (Marine phytoplankton as biogeochemical drivers: Scaling from membranes and single cells to populations)
Période du rapport: 2020-04-01 au 2020-09-30
The single-celled photosynthetic eukaryotic phytoplankton fix inorganic carbon into organic molecules that form the building blocks of life. They form the basis of the marine food web on which all marine life depends. The phytoplankton also drive other major processes in the ocean such as calcification by coccolithophores and silicification by diatoms. We are beginning to gain exciting new insights into the mechanisms and evolution of membrane transport, cell signalling and metabolic regulation in phytoplankton. The SeaCells project brings together single cell biophysics, imaging and state of the art molecular biology to addresses fundamental questions in phytoplankton biology from cellular to population scales. A significant aim is to gain critical mechanistic understanding at the molecular and single cell level along with information on the microenvironment that surrounds cells. In order to understand how the physiological properties of single cells in the laboratory translate to behaviour of natural populations we will transfer single cell technologies developed in the laboratory to ship-board platforms. The project also aims to understand how the variability in the responses of individual phytoplankton cells to environmental perturbations is likely to determine the overall effects of changing ocean conditions on natural phytoplankton populations.
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.
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.
1. New approaches in microscopy. The Mesoscope (www.Mesolens.com) presents for the first time a high resolution imaging system with a very wide field of view. We have shown that it is possible to gain sub-cellular fluorescence resolution of every cell in a population of more than 10,000 cells. This system offers a number of advantages over current fluorescence and confocal systems for monitoring both laboratory cultures and natural populations. The system is now fully operational and is allowing the first in depth studies of single cell physiological and signalling processes in large populations of cells.
We have had much interest in this system from other biological oceanographers who are keen to explore the application of this system for monitoring individual cell behaviour in situ in large populations of phytoplankton. By applying this new technology to laboratory cultures, freshly isolated natural samples and natural populations in situ we expect that this approach will provide much novel information on the responses of marine plankton to changes in climate and ocean chemistry.
2. A new class of eukaryotic ion channels. Our discovery of single-domain cation channels in diatoms and coccolithophores sheds new light on the evolution of ion channels. It also provides new models for the elucidation of voltage dependent cation channel function and regulation. We have proposed that these novel channels most likely bear similarities to primitive ancestral ion channels from which the 4-domain voltage dependent cation channels evolved in animals. Progress in this work has been prepared for publication. We will continue to characterise the functional roles of this new class of eukaryotic cation channels using both in vivo reporter and heterologous expression approaches.
3. New mechanistic understanding of ocean acidification impacts on coccolithophores. We have uncovered a key regulatory mechanism that becomes impaired when coccolithophores are acclimated to lowered pH. This also leads to specific malformations in coccolith morphology. Surprisingly, we found that population responses are the result of very different responses between individual cells in the population, providing new insights into how individuals determine the overall acclimation response of a population.
We have had much interest in this system from other biological oceanographers who are keen to explore the application of this system for monitoring individual cell behaviour in situ in large populations of phytoplankton. By applying this new technology to laboratory cultures, freshly isolated natural samples and natural populations in situ we expect that this approach will provide much novel information on the responses of marine plankton to changes in climate and ocean chemistry.
2. A new class of eukaryotic ion channels. Our discovery of single-domain cation channels in diatoms and coccolithophores sheds new light on the evolution of ion channels. It also provides new models for the elucidation of voltage dependent cation channel function and regulation. We have proposed that these novel channels most likely bear similarities to primitive ancestral ion channels from which the 4-domain voltage dependent cation channels evolved in animals. Progress in this work has been prepared for publication. We will continue to characterise the functional roles of this new class of eukaryotic cation channels using both in vivo reporter and heterologous expression approaches.
3. New mechanistic understanding of ocean acidification impacts on coccolithophores. We have uncovered a key regulatory mechanism that becomes impaired when coccolithophores are acclimated to lowered pH. This also leads to specific malformations in coccolith morphology. Surprisingly, we found that population responses are the result of very different responses between individual cells in the population, providing new insights into how individuals determine the overall acclimation response of a population.