Soil zymography: a decade in microbial hotspot imaging and future challenges

Soil zymography represents a non-invasive methodology facilitating the in situ visualization and localization of potential enzyme activities in soil. Due to universality and simplicity of zymography method, it has been widely used to visualize the hidden microbial and root life in soil, which is a very heterogeneous and “dark” environment. Following the pioneering use of fluorogenic substrates for enzyme activity visualization in soil, a significant methodological advancement occurred within the subsequent decade. Our primary focus is to highlight the progress in the last 10 years, with respect to the spectrum of enzyme activity imaging, zymography resolution, standardization and hotspot identification. Specifically, we emphasize the integration of zymography with the visualization of soil acidification, root exudation, pore distribution, nutrient and water movements. Although the majority of applications so far have centered on enzyme activities in the rhizosphere to reveal plant-soil-microbial interactions, we present the case studies to identify soil heterogeneity and microbial activity also in biopores, detritusphere and other hotspots. We present not only the advancements made but also the current possibilities, challenges, and the potential directions of soil zymography. This technique finds applications across natural and agricultural ecosystems, both in field settings and laboratory studies, capable of scaling from the entire root system (dm) down to microbial communities (μm). In the decade ahead, the future of enzyme research, particularly zymography imaging, will continue to broaden the scope of microbial studies and hotspot localization. This expansion will involve integrating with disciplines such as (bio)chemical, physico-chemical, microbial cell imaging, and isotope applications, facilitating a deeper understanding of soil processes and microbial interactions.