Extracellular phosphatase activity (PA) has been used as an overall indicator of P depletion in lake phytoplankton. However, detailed insights into the mechanisms of PA regulation are still limited, especially in the case of acid phosphatases. The novel substrate ELF97 phosphate allows for tagging PA on single cells in an epifluorescence microscope. This fluorescence-labeled enzyme activity (FLEA) assay enables for autecological studies in natural phytoplankton and algal cultures. We combined the FLEA assay with image analysis to measure cell-specific acid PA in two closely related species of the genus Coccomyxa (Trebouxiophyceae, Chlorophyta) isolated from two acidic lakes with distinct P availability. The strains were cultured in a mineral medium supplied with organic (beta-glycerol phosphate) or inorganic (orthophosphate) P at three concentrations. Both strains responded to experimental conditions in a similar way, suggesting that acid extracellular phosphatases were regulated irrespectively of the origin and history of the strains. We found an increase in cell-specific PA at low P concentration and the cultures grown with organic P produced significantly higher (ca. 10-fold) PA than those cultured with the same concentrations of inorganic P. The cell-specific PA measured in the cultures grown with the lowest organic P concentration roughly corresponded to those of the original Coccomyxa population from an acidic lake with impaired P availability. The ability of Coccomyxa strains to produce extracellular phosphatases, together with tolerance for both low pH and metals can be one of the factors enabling the dominance of the genus in extreme conditions of acidic lakes. The analysis of frequency distribution of the single-cell PA documented that simple visual counting of 'active' (labeled) and 'non-active' (non-labeled) cells can lead to biased conclusions regarding algal P status because the actual PA of the 'active' cells can vary from negligible to very high values. The FLEA assay using image cytometry offers a strong tool in plankton ecology for exploring P metabolism.

• Keywords
• Coccomyxa, ELF97 phosphate, FLEA technique, acid phosphatase, image cytometry, inorganic phosphorus, organic phosphorus, phosphorus limitation,
• Publication type
• Journal Article MeSH

Extracellular phosphatase production by phytoplankton was investigated in the moderately eutrophic Lipno reservoir, Czech Republic during 2009 and 2010. We hypothesized that production of extracellular phosphatases is an additional mechanism of phosphorus acquisition enabling producers to survive rather than to dominate the phytoplankton. Hence, we examined the relationship between light availability and phosphatase production, as light plays an important role in polymictic environments. Bulk phosphatase activity was measured using a common fluorometric assay, and the production of phosphatases was studied using the Fluorescently Labelled Enzyme Activity technique, which enabled direct microscopic detection of phosphatase-positive cells. In total, 29 taxa of phytoplankton were identified during both years. Only 17 taxa from the total number of 29 showed production of extracellular phosphatases. Species dominating the phytoplankton rarely produced extracellular phosphatases. In contrast, taxa exhibiting phosphatase activity were present in low biomass in the phytoplankton assemblage. Moreover, there was a significant relationship between the proportion of phosphatase positive species in samples and the Z(eu):Z(mix) ratio (a proxy of light availability). A laboratory experiment with different light intensities confirmed the influence of light on production of phosphatases. Our seasonal study confirmed that extracellular phosphatase production is common in low-abundance populations but not in dominant taxa of the phytoplankton. It also suggested the importance of sufficient light conditions for the production of extracellular phosphatases.

• MeSH
• Phosphoric Monoester Hydrolases biosynthesis   MeSH
• Phytoplankton enzymology   radiation effects   MeSH
• Light * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphoric Monoester Hydrolases MeSH

• Here, enzymatic activity of five hydrolases was measured fluorometrically in the fluid collected from traps of four aquatic Utricularia species and in the water in which the plants were cultured. • In empty traps, the highest activity was always exhibited by phosphatases (6.1-29.8 µmol l-1 h-1 ) and β-glucosidases (1.35-2.95 µmol l-1 h-1 ), while the activities of α-glucosidases, β-hexosaminidases and aminopeptidases were usually lower by one or two orders of magnitude. Two days after addition of prey (Chydorus sp.), all enzymatic activities in the traps noticeably decreased in Utricularia foliosa and U. australis but markedly increased in Utricularia vulgaris. • Phosphatase activity in the empty traps was 2-18 times higher than that in the culture water at the same pH of 4.7, but activities of the other trap enzymes were usually higher in the water. Correlative analyses did not show any clear relationship between these activities. • Trap comensals (Euglena) could be partly responsible for production of some trap enzymes. The traps can produce phosphatases independently of catching prey. Taking into account the enzymatic activities in traps, phosphorus uptake from prey might be more important than that of nitrogen for the plants.

• Keywords
• aminopeptidase, aquatic carnivorous plants, chitinase, extracellular enzymatic activity, glucosidase, phosphatase, trap fluid pH, β-hexosaminidase,
• Publication type
• Journal Article MeSH