Keep oxygen in check: An improved in-situ zymography approach for mapping anoxic hydrolytic enzyme activities in a...

Paddy soils regularly experience redox oscillations during the wetting and draining stages, yet the effects of short-term presence of oxygen (O₂) on in-situ microbial hotspots and enzyme activities in anoxic ecosystems remain unclear. To fill this knowledge gap, we applied soil zymography to localize hotspots and activities of phosphomonoesterase (PME), β-glucosidase (BG), and leucine aminopeptidase (LAP) in three compartments of rice-planted rhizoboxes (top bulk, rooted, and bottom bulk paddy soil) under oxic (+O₂) and anoxic (O₂) conditions. Short-term (35 min) aeration decreased PME activity by 13-49 %, BG by 4-52 %, and LAP by 12-61 % as compared with O₂ in three soil compartments. The percentage of hotspot area was higher by 3-110 % for PME, by 10-60 % for BG, and by 12-158 % for LAP under +O₂ vs. O₂ conditions depending on a rice growth stage. Irrespective of the aeration conditions, the rhizosphere extent of rice plants for three enzymes was generally greater under higher moisture conditions and at earlier growth stage. Higher O₂ sensitivity for the tested enzymes at bottom bulk soil versus other compartments suggested that short-term aeration during conventional zymography may lead to underestimation of nutrient mobilization in subsoil compared to top bulk soil. The intolerance of anaerobic microorganisms against the toxicity of O₂ in the cells and the shift of microbial metabolic pathways may explain such a short-term suppression by O₂. Our findings, therefore, show that anoxic conditions and soil moisture should be kept during zymography and probably other in-situ soil imaging methods when studying anoxic systems.