Primary production in the world ocean sustains marine organisms and modulates global climate. As a result, the factors that regulate phytoplankton production are a source of extensive investigation. Much attention focuses on the role of nutrients, such as nitrogen and phosphorus, in the regulation of phytoplankton growth and bloom dynamics. However, studies have been hampered by the difficulties involved in assessing single-cell phytoplankton physiology in situ. This research discusses the development of two in situ assays for phosphate stress in the dinoflagellate Prorocentrum minimum. This work also elucidates aspects of phosphate nutrition in this species and the coccolithophorid Emiliania huxleyi.

For both organisms cell-surface proteins regulated by phosphate were characterized in laboratory cultures. The induction and repression of these proteins in response to phosphate supply were studied and compared with alkaline phosphatase activity which is a commonly used marker of phosphate stress. In P. minimum one phosphate regulated protein was purified and identified as an alkaline phosphatase. The phosphate regulated-proteins of E. huxleyi were partially purified, consistently associating with each other as well as with alkaline phosphatase activity. One of these proteins appears to be a phosphatase with an affinity for 5' nucleotides.

Antibody probes were generated to the purified alkaline phosphatase in P. minimum. The antibody probes were tested for specificity to the target protein and for cross reactivity with other species of phytoplankton. An immunofluorescence assay using these antibody probes distinguished phosphate-stressed from phosphate-replete cells in culture and in a field sample.

In P. minimum an additional assay for phosphate stress was developed using the fluorescent alkaline phosphatase substrate ELF-97. The phytoplankton population in Narragansett Bay, Rhode Island was sampled in the summer of 1998 and tested for phosphate stress using this assay. Cell-specific, P. minimum alkaline phosphatase activity was detected in the field samples. This study demonstrates the importance of phosphate in aspects of phytoplankton physiological ecology, particularly in this important estuary. It also confirms that the tools developed here may be used successfully in future studies of cell-specific phytoplankton physiological ecology.