The key difference between the membrane
The key difference between the membrane zymography and classical measurements of enzyme activities in soil slurries is the enzyme-substrate accessibility. Classical enzyme assays maximize access of substrate to all potentially reactive enzyme sites by ensuring sample destruction and detachment followed by the release of enzymes from soil matrix to the suspension (Schimel et al., 2017). However, in intact soil, an occurrence of a contact between a substrate and an enzyme depends on the presence of transport avenues and on Phos-tag processes taking place within them. Membrane zymography to a certain extent emulates the diffusion processes taking place in the intact soil. Just as in soil, the fluorescent patterns on the membrane depend on diffusion. This feature is both a blessing and a curse of the method, as it enables clear visualization of the real processes occurring in soil, but substantially complicates interpretation of its results. The activity detected in a course of soil zymography is generally attributed to extracellular enzymes excreted by roots or microorganisms, which exist either immobilized on surfaces of the soil matrix and organic materials or in a free form in the soil solution (Gianfreda and Bollag, 1994; Rao et al., 2000). The immobilized enzymes are assumed to be strongly attached, thus immobile, while the free enzymes are assumed to be mobile (Nannipieri and Gianfreda, 1998). While it is presumed that both forms exist (Stotzky, 1986; Nannipieri et al., 1996), their relative sizes are generally unknown and likely dynamic, due to fluctuations in enzyme production by roots and microorganisms, and continuous biochemical enzyme degradation and their immobilization by soil particles and organic material. The soil surface in contact with zymography membrane is, typically, very uneven. Thus, as the distances between enzymes located on/near the soil surface and the membrane increase, the potential contributions of enzymes to the fluorescent signal on the membrane decrease (Fig. 1). Free enzymes in the soil solution can potentially reach the membrane by diffusion or react with dissolved substrate within the water film boundary between the membrane and soil surface. Immobilized enzymes can be broadly divided into three groups in terms of their position with respect to the membrane: 1) enzymes in direct physical contact with the membrane, 2) enzymes in hydrological contact with the membrane through water films, and 3) enzymes without any contact with the membrane. When the membrane with a substrate is placed on a soil surface, the enzymes in direct contact with the membrane (group 1) are the first to be involved in catalytic activities. The substrate readily available for this enzyme group is decomposed quickly and the released MUF immediately contributes to appearance of a fluorescent signal on the membrane. Involvement in catalyzes of the enzymes connected with the membrane through water films (group 2) depends on the time needed, first, for the substrate to diffuse from the membrane and reach the enzymes, and, second, for the released MUF to diffuse back to the membrane. The immobilized enzymes with no contact with the membrane (group 3) are unlikely to contribute to the fluorescent signal, since there are no means for the substrate and MUF transport to/from them (Fig. 1). Thus, the fluorescent signal detected on the membrane under UV-light may originate from multiple sources including immobilized and free enzymes. Upon reaching the membrane, the MUF products can also laterally diffuse within it during the incubation and thus decrease the spatial resolution of the zymogram. Higher catalytic activity of free enzymes as compared to immobilized ones (Rao et al., 2000) further complicates interpretation of soil zymograms. The same complications exist in standard fluid-based enzyme assays. It should be noted that diffusion, in contrast to convective flow, is omnidirectional, and rates of either vertical or lateral diffusion of substances, enzymes, and products, e.g. MUF, in soils and membranes are generally unknown. Therefore, quantification of in situ enzyme activity in the soil and the rhizosphere, based on membrane zymography, requires accurate assessments of diffusion pathways and rates for all involved chemicals. We assessed following diffusion pathways involved in zymography analysis: 1) diffusion of enzymes from soil into zymography membranes; 2) diffusion of MUF and substrate within the membranes; 3) diffusion of substrate from the membrane to soil; and 4) diffusion of MUF from soil to the membrane.