Enzyme-based visual indicators of organic nitrogen transformation: Sensitivity to mineral fertilization in the maize rhizosphere

Interplay between mineral and organic nitrogen (N) in the rhizosphere is driven by root-part-specific traits that modulate spatially distinct microbial response to nutrient limitation. Co-localization of organic-N and related hydrolytic enzymes requires visual identification of organic N hubs and enzyme-mediated amino-N transformation, yet the sensitivity of visualization techniques needs to be compared with common microplate-based enzymatic assays. Although ecoenzymatic stoichiometry is widely used to assess microbial nutrient limitation, its applicability at the microscale of heterogeneous organic N hubs needs validation. We integrated amino-mapping, leucine aminopeptidase (LAP) zymography, and microplate enzymatic assays to quantify organic N distribution in maize ( Zea mays L.) rhizosphere under full and reduced mineral fertilization. We evaluated the sensitivity of LAP-to-amino-N ratio as a process-based indicator of amino-N transformation intensity and assessed its complementarity with ecoenzymatic stoichiometric models. Amino-N hubs and LAP activity were mainly associated with root tips and seminal roots. The rhizosphere of lateral roots, however, was depleted in amino-N despite intensive LAP activity. Root parts showed distinct compensatory responses to reduced fertilization. In seminal tips rhizosphere, 1.8-2.9 times elevated LAP activity under reduced fertilization indicated a nutrient-mining strategy. In seminal roots and lateral tips rhizosphere, 22-51% larger amino-N content under reduced versus full fertilization suggested organic N retention as a buffering mechanism. Ecoenzymatic stoichiometric models based on microplate assay unexpectedly indicated microbial N limitation under full fertilization. Such a “limitation” was aligned with a lower amino-N content and higher LAP activity in the dominant area of the seminal roots and lateral tips. This indicated an intensive transformation of organic N that could potentially cause local N deficiency. The LAP-to-amino-N ratio increased in seminal tips but declined in seminal roots and lateral tips under reduced versus full fertilization, providing complementary interpretations of root-part-specific responses to fertilization. This study supplied spatially resolved evidence that root-part-specific traits and fertilization shaped organic N transformation, highlighting the importance of integrating visualization-based approaches with bulk enzyme assays to disentangle fine-scale N cycling processes.