Razavi, B. S., X. Zhang, N. Bilyera, A. Guber, and M. Zarebanadkouki. 2019. Soil zymography: Simple and reliable? Review of current knowledge and optimization of the method. Rhizosphere 11:100161.
Within just a few years, soil zymography has become accepted as an attractive and unique approach for 2D mapping of enzyme activities in intact soil samples. With zymography, enzymatic conversion of the substrate into a hydrolysis reaction product can literally be visualized. Soil zymography is, however, fraught with methodical difficulties due to: (i) membrane or gel attachment to the soil surface; (ii) diffusion of substrates through the membrane or gel and of reaction products back to the membrane; (iii) strong effect of imaging (photography) and image analysis on the results. In this review, we describe important procedural details of soil zymography and define the steps necessary to properly visualize enzyme activities in environmental samples. We make the following recommendations to improve zymography results 1) run soil background imaging prior to any soil zymography; 2) confirm that roots are in the soil and not on top of the soil surface; 3) perform soil zymography under the initial environmental conditions of the samples (temperature, water content, light intensity, etc); 4) examine whether membrane/gel attachment during the incubation is appropriate to properly measure enzyme activity; 5) find the right balance between saturating substrate concentration of soil and selected substrate concentration for zymography; 6) run proper standards to ensure that enzyme activity values can be accurately calculated; 7) fix camera settings and photography conditions; 8) ensure that images are properly analyzed. These steps should help to develop a unified visualization of enzyme activities in soil and ecosystem ecology. Finally, coupling of soil zymography with other imaging techniques and advanced analytical approaches will give insight into the net effect of multiple processes, such as root respiration, rhizodeposition, nutrient and metal(loid) dynamics, plant-mediated oxygen release, microbial respiration and reoxidation of reduced compounds in relation to the activities of enzymes released by plants or microbes.
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