Detection and quantification of alkaline phosphatase in single cells of phosphorus-starved marine phytoplankton

Abstract

Alkaline phosphatase (AP) activity in marine and freshwater phytoplankton has been associated with phosphorus (P) limitation whereby the enzyme functions in the breakdown of exogenous organic P compounds to utilizable inorganic forms. Current enzyme assays to determine the P status of the phytoplankton measure only the AP activity of the whole community and do not yield information on individual species. A new insoluble fluorogenic substrate for AP, termed ELF (Enzyme-Labeled Fluorescence), yields a stable, highly fluorescent precipitate at the site of enzyme activity and thus has the capability to determine the P status of individual cells. In this study, ELF was utilized for in situ detection and quantification of AP in marine phytoplankton cultures and a comparison was made between the insoluble ELF substrate and several soluble AP substrates [3-O-methylfluorescein phosphate (MFP), 3,6-fluorescein diphosphate (FDP) and Attophos]. Non-axenic batch cultures of Alexandrium fundyense, Amphidinium sp. and Isochrysis galbana were grown in different media types using orthophosphate as an inorganic source and sodium-glycerophosphate as an organic source, with final phosphate concentrations ranging from 38.3 to 3 μM (i.e. f/2, f/40, f/80, plus ambient P). Epifluorescence microscopy was used to determine if and where 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 reader (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 Amphidinium sp. and I. galbana cells, but was seen in some A. fundyense cells only during the late stationary phase. Cells grown in high phosphate concentrations (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 with the precipitable substrate. Similar results were obtained when the cultures were analyzed by flow cytometry. Except for A. fundyense, cells 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 analysis using the soluble substrates MFP, FDP, and Attophos(TM) on the Cytofluor showed little activity for A. fundyense, but high fluorescence for P-starved Amphidinium sp. and I. galbana. Insoluble ELF thus provides a means to detect and quantify AP in individual cells using visual observations or flow cytometry. This technique offers a new level of resolution and sensitivity at the single cell level that can provide insights into the P nutrition of phytoplankton and other microorganisms in natural waters.