Aims Visualization of enzymatic activity links microbial functioning to localization in heterogeneous soil habitats. To assess enzymatic reactions in soil thin layer at the microscopic level, we developed a micro-zymography approach and tested it by visualization of the potential activity of phosphomonoesterase for aggregates collected from the rhizosphere of Zea mays L. Methods We evaluated micro-zymography by applying fluorogenically-labeled substrate i) on individual soil aggregates freshly sampled from the rhizosphere, ii) on thin layers of aggregates (≈ 500 µm) saturated with substrate to assess the dynamics of phosphomonoesterase activity, and iii) on maize roots under laser scanning microscope upon the identified hotspots by membrane-based zymography. Results We found super transparent silicon as the most appropriate fixative to prevent sample drying. We optimized microscope settings to eliminate the soil auto-fluorescence. The fluorescent signal shifted from the free liquid phase towards the aggregate boundaries within 30 min after substrate addition and was finally detectable at the surface of a few aggregates. This was probably due to higher microbial abundance and enzymatic activity on the soil aggregates compared to the liquid phase. The enzymatic activity appeared patchy at the aggregate and root surfaces indicating heterogeneous distribution of hotspots. Conclusions The methodology including calibration, sample preparation, fixation, and monitoring was developed. The novel membrane-free micro-zymography approach is a promising tool to identify functional specificity and niche differentiation on roots and soil aggregates. This approach revealed unexplained complexity of competing processes (biochemical, hydrolytic, and physical) due to differently charged reaction products and enzyme-clay complexes.
<p>Enzymes are produced by microorganisms either intracellularly in cell&#8217;s cytoplasm and periplasm or extracellularly either as attached to outer surface of cell membranes or released to the soil microhabitats. The distribution of microhabitats in soil is highly heterogeneous with high abundance of microorganisms in the small volume of soil hotspots, e.g., in the rhizosphere - the most important plant-soil interface with very dynamic interactions between roots and microorganisms. Soil zymography is one of the most realistic methods developed to visualize enzyme activity in undisturbed soil at the mesoscale (mm-cm) level using substrate-saturated membranes. However, visualization of enzymatic processes at the <strong>micro</strong>-scale level remains a challenge. We tested several impregnation strategies of soil sample (e.g., by agarose gel, silicon spray and super transparent silicon mixture) for their suitability for micro-zymography, i.e., for visualization of enzyme activity in undisturbed soil particles at the microscopic level combining fluorogenic substrates with epifluorescence microscopy. The pros- and cons- of various combinations of impregnation and staining of micro-sized soil samples will be discussed.</p><p><strong>Keywords:</strong> enzyme activity, zymography, fluorogenic substrates</p>
<p>As N limitation strongly influences ecosystem functioning, numerous studies explored the transformation process of mineral nitrogen. In contrast, the importance of organic nitrogen, which can short-circuit the mineralization step, for plant nutrition in different ecosystems often overlooked. A spatial link between the sources of organic N and N-acquiring enzymatic activity in soil is still missing due to the lack of suitable techniques. Here we developed a novel approach: <em>in situ</em> amino-mapping and coupled it with time-lapse zymography to quantify distribution of organic nitrogen in the rhizosphere of <em>Zea mays</em> L and tested spatial association of enzymatic activity with organic nitrogen abundance at the root-soil interface. Coupling the two approaches enabled identification the hotspots of amino-N, and revealed their co-occurrence with N-related enzymatic activity in seminal roots and root tips: intensive enzymatic activity was accompanied by large amino-N content, especially in the rhizosphere of seminal root tips. This work was conducted within the framework of the Priority program 2089 &#8220;Rhizosphere spatiotemporal organization &#8211; a key to rhizosphere functions&#8221;, funded by German Research Foundation (DFG &#8211; Project number: 403664478). Seeds of the maize were provided by Caroline Marcon and Frank Hochholdinger (University of Bonn).</p>
<p>Alive plants and soil microorganisms are the influential sources of extracellular enzymes facilitating decomposition of polymeric organic compounds. Enzyme activities are especially intensive and spatially heterogeneous in the rhizosphere, where microorganisms are stimulated by rhizodeposition. Two-dimensional activity distribution of hydrolytic enzymes participating in transformation of soil organics in the distance gradients from the root can be visualized under UV light by zymography - by placing a fluorogenic substrate-saturated membrane on the soil surface. Functional traits of enzymes can be co-localized with spatial distribution of enzymatic activity by precise micro-sampling based on zymography. We used rhizobox experiment to visualize activity of &#946;-glucosidase, leucine aminopeptidase, and phosphatase in the rhizosphere of wild type and root hairless mutant of <em>Zea mays</em> L. cultivated for 3 weeks. After precise micro-sampling, we determined kinetic parameters of enzymes: max potential activity and affinity to substrate in the rhizosphere gradients. Finally, we compared the correspondence of enzymatic activity determined by zymography and by kinetic approach. This work was conducted within the framework of the priority program 2089, funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) &#8211; Project number: 403664478. Seeds of the maize were provided by Caroline Marcon and Frank Hochholdinger (University of Bonn).</p><p><strong>Keywords:</strong> zymography, enzyme kinetic, maize, rhizosphere gradients</p>
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