Time-lapse approach to correct deficiencies of 2D soil zymography

Membrane zymography is commonly used in rhizosphere ecology to map enzyme activities in intact soil samples and plant roots. The method consists of incubating a membrane saturated with an enzyme-specific fluorogenic substrate on the soil/root surface followed by measurements of fluorescence intensity of the product in the membrane. The traditional zymography is based on assumptions that fluorescence on membrane images is linearly increasing with time during zymography and an increase in product content is numerically equal to enzyme activity in soil below the membrane. These assumptions are unlikely to hold in experimental settings. Here we introduce a new zymography technique, time-lapse zymography (TLZ); the approach that eliminates the need for assumptions of the traditional zymography and provides more realistic estimates of enzymatic activities. We assessed the new technique in a series of laboratory and modeling experiments, including quantification of the fluorescent product diffusion (e.g. MUF: 4-methylumbelliferone) from plant roots, enzyme activity measurements on the roots, and HYDRUS-2D & HP2 software calibration with obtained data. The calibrated model was used to analyze the processes governing spatial and temporal dynamics of MUF contents in the membrane. The results indicated that the enzyme diffusion within the membrane-soil system was negligible, and measured zymograms were adequately reproduced solely by accounting for substrate and product diffusions and for catalytic enzyme reaction described by the Michaelis-Menten equation. TLZ enabled identifying and using linear parts on MUF time series and accounting for MUF losses in each zymogram pixel, considerably improving the accuracy as compared to the traditional method. Results demonstrated that enzymatic activity from only a thin soil layer (similar to 0.2 mm) is adequately represented in zymograms.