An Experimental Insight into Extracellular Phosphatases – Differential Induction of Cell-Specific Activity in Green Algae Cultured under Various Phosphorus Conditions

Abstract

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.

Bacterial Recruitment to Carnivorous Pitcher Plant Communities: Identifying Sources Influencing Plant Microbiome Composition and Function

“…For chitinase and protease, the early stimulation of activity followed by a slow decline over several weeks ( Figure 6 ) is consistent with changes over time in field pitchers ( Grothjan, 2021 ). In contrast, phosphatase activity increased over time in artificial pitchers, probably related to the growth of green algae which produce abundant extracellular phosphatase activity ( Young et al, 2010 ; Vrba et al, 2018 ). Aeromonadaceae , which were only observed in high abundance in artificial pitchers ( Figure 2 ) are also known for producing alkaline phosphatase ( Ghosh and Homechaudhuri, 2011 ).…”

Strategies adopted by Aphanizomenon flos-aquae in response to phosphorus deficiency and their role on growth

“…S4). Consistently, the Coccomyxa isolated from Lakes Plesne and Hromnice in Czechia increased in cell-specific phosphatase activity measured combining the ELF assay with image analysis at low P concentration and the cultures treated with DOP (β-glycerol phosphate), which generated remarkably higher activity than those supplied with the same Pi concentrations [55]. Therefore, APase in A. flosaquae could perform its function of hydrolyzing organic P, as illustrated by significant and positive relationship between the ELFp and APA >3.0μm (Additional file 1: Fig.…”

Strategies adopted by Aphanizomenon flos-aquae in response to phosphorus deficiency and their role on growth

“…S4). Consistently, the Coccomyxa isolated from Lakes Plesne and Hromnice in Czechia increased in cell-specific phosphatase activity measured combining the ELF assay with image analysis at low P concentration and the cultures treated with DOP (β-glycerol phosphate), which generated remarkably higher activity than those supplied with the same Pi concentrations [56]. Therefore, APase in A. flos-aquae could perform its function of hydrolyzing organic P, as illustrated by significant and positive relationship between the ELFp and APA >3.0μm (Fig.…”