Biochar Modulates Leucine Aminopeptidase Hotspots and Kinetics under Salinity Stress in the Wheat Rhizosphere

Salinity stress is a major factor limiting microbial activity in soils, as it can impair enzymatic processes by destroying microbial cells and disrupting root exudation. In contrast, biochar, through the improvement of soil physicochemical properties, may stimulate microbial growth and functionality. However, the response of soil enzymes to the simultaneous presence of biochar and salinity stress has been scarcely investigated. In the present study, we assessed the effects of two salinity levels (0 and 150 mM NaCl) under two biochar treatments (0 and 2%) on spatial distribution and kinetic parameters of leucine aminopeptidase (LAP) activity in the rhizosphere of wheat by combining zymography with enzyme kinetics.

In the presence and absence of biochar, salinity reduced the hotspots of LAP activity by 15.8% and 15.7% compared to the respective control, respectively. In contrast, at both biochar levels, salinity increased the rhizosphere extent of LAP compared to the respective control. Biochar nearly doubled hotspots of LAP activity compared to its absence, yet it simultaneously reduced the rhizosphere extent of LAP at both salinity levels. Generally, the highest LAP activity hotspots and the lowest rhizosphere extent of LAP were observed in the of 2% biochar treatment under non-saline condition. The analysis of enzyme kinetics (V_max, K_m) in the hotspots showed salinity caused an increase in enzyme affinity for substrate (K_m decreased by 37.9% to 97.2%) at both levels of biochar. In contrast, biochar decreased enzyme affinity for the substrate (as indicated by a 1.1- to 2.2-fold increase in K_m) under both salinity levels. Biochar increased potential enzymatic activity (V_max) in the hotspots, reaching 1.9 times higher than without biochar. Conversely, under salinity conditions, this activity decreased relative to optimal conditions at both biochar levels. Overall, the 2% biochar treatment under non-saline condition showed the highest V_max and K_m, whereas the non-biochar treatment under saline condition indicated the lowest.

These patterns collectively indicate that salinity and biochar exert contrasting controls on rhizosphere enzymatic functioning by modifying both microbial physiology and microhabitat conditions. Salinity imposes physiological stress and reduces root-derived substrates, driving microbial communities toward more dispersed activity and the production of high-affinity enzymes optimized for resource scarcity. In contrast, biochar enhances microhabitat quality, stimulating microbial activity and catalytic capacity despite reducing enzyme affinity, likely due to changes in community composition or enzyme–biochar interactions. Overall, biochar strengthens rhizosphere functioning but cannot fully offset the inhibitory effects of salinity on microbial metabolism and enzymatic efficiency.