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

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), ß-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.