Enzymatically hydrolyzed fluorescence-based chemical probe enables in situ mapping of chitinase activity in the rhizosphere

Chitin is an insoluble and ubiquitous soil biopolymer, estimated to be the second most abundant organic soil biopolymer on Earth. Despite its abundance, role as a source of C and N in soil, and importance to ecosystem function, further research is required to elucidate key controls on chitin breakdown under varying environmental conditions. Previous work highlights the important role rhizosphere microbiomes and root exudates can play in chitin catabolism. To enable mapping of chitinase activity within the highly heterogeneous and spatially organized rhizosphere, we designed and synthesized an enzymatically activated fluorogenic substrate, chitotriose-TokyoGreen (chitotriose-TG), by incorporating a fluorescein derivative (TG) onto the trimeric unit of chitin. This non-fluorescent substrate is selectively hydrolyzed by chitinase to release TG and yield a fluorescence signal, which can be used to spatially image and measure chitinase activity in the rhizosphere. To demonstrate the application of this technique, we grew switchgrass (Panicum virgatum) in rhizoboxes amended with a horizontal layer supplemented with chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offers non-destructive enzyme extraction while preserving the 2D spatial position of the enzymes. We then subjected these membranes to the synthesized chitotriose-TG stain to spatially visualize the distribution of chitinase activity within the rhizosphere. We observed increased chitinase activity near switchgrass roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase production with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can enhance efforts to better understand spatial controls on chitin breakdown in rhizosphere, further elucidating the role of chitin as a C and N source in these systems.