DETECTION AND QUANTIFICATION OF ALKALINE-PHOSPHATASE IN SINGLE CELLS OF PHOSPHORUS-STARVED MARINE-PHYTOPLANKTON

Alkaline phosphatase (AP) activity in marine and freshwater phytoplank ton has been associated with phosphorus (P) limitation whereby the enz yme functions in the breakdown of exogenous organic P compounds to uti lizable inorganic forms. Current enzyme assays to determine the P stat us of the phytoplankton measure only the AP activity of the whole comm unity and do not yield information on individual species. A new insolu ble fluorogenic substrate for AP, termed ELF (Enzyme-Labeled Fluoresce nce), yields a stable, highly fluorescent precipitate at the site of e nzyme activity and thus has the capability to determine the P status o f individual cells. In this study, ELF was utilized for in situ detect ion and quantification of AP in marine phytoplankton cultures and a co mparison was made between the insoluble ELF substrate and several solu ble AP substrates [3-O-methylfluorescein phosphate (MFP), 3,6-fluoresc ein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alex andrium fundyense, Amphidinium sp. and Isochrysis galbana were grown i n different media types using orthophosphate as an inorganic source an d sodium-glycerophosphate as an organic source, with final phosphate c oncentrations ranging from 38.3 to 3 mu M (i.e, f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and wh ere the cells were labeled with ELF, while flow cytometry was used to quantify the amount of ELF retained on individual cells. The detection of the soluble substrates utilized a multiwell fluorescence plate rea der (Cytofluor(TM)). Only cells grown in low phosphate concentrations (f/40, f/80) exhibited the bright green fluorescence signal of the ELF precipitate. This signal was always observed for P-starved Amphidiniu m sp, and I. galbana cells, but was seen in some A. fundyense cells on ly during the late stationary phase. Cells grown in high phosphate con centrations (i.e. at f/2 levels) showed no ELF fluorescence. Slightly positive soluble substrate assays suggest that these species may have produced small amounts of AP constitutively that were not detected wit h the precipitable substrate. Similar results were obtained when the c ultures were analyzed by flow cytometry. Except for A, fundyense, cell s grown in low phosphate concentrations showed high ELF fluorescence. However, no positive ELF fluorescence was detected with the Cytofluor for all 3 species due to lack of instrument sensitivity. Comparable an alysis using the soluble substrates MFP, FDP, and Attophos(TM) on the Cytofluor showed little activity for A. fundyense, but high fluorescen ce for P-starved Amphidinium sp. and I. galbana. Insoluble ELF thus pr ovides a means to detect and quantify AP in individual cells using vis ual observations or now cytometry. This technique offers a new level o f resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganis ms in natural waters.