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

Journal Article · · Soil Biology and Biochemistry
 [1];  [2];  [2];  [2];  [2];  [3];  [4]
  1. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Washington State Univ., Pullman, WA (United States)
  2. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
  3. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Washington State Univ., Pullman, WA (United States); Baylor Univ., Waco, TX (United States)
  4. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States); Michigan State Univ., East Lansing, MI (United States); Washington State Univ., Pullman, WA (United States)
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Rhizosphere microbiomes and root exudates play a pivotal biochemical role in helping to catalyze chitin catabolism. Chitin is a recalcitrant 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, the biochemical mechanisms controlling chitin fate in the rhizosphere are elusive and poorly understood. To enable spatial mapping of chitinase activity in the 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 high 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 of chitin. We extracted mobile proteins from the rhizobox using a nitrocellulose membrane blotting technique which offer 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. Furthermore, we observed increased chitinase activity near plant roots and higher activity within the soil zone enriched in chitin, showing an adaptive response of chitinase activity with spatial focusing in areas of higher chitin abundance. Thus, the enzyme extraction and visualization strategy we describe here can help enlighten 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.

Research Organization:
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
Grant/Contract Number:
AC05-76RL01830
OSTI ID:
2000746
Report Number(s):
PNNL-SA--182442
Journal Information:
Soil Biology and Biochemistry, Journal Name: Soil Biology and Biochemistry Vol. 184; ISSN 0038-0717
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Chitin
Chitinase
Fluorescence probe
Rhizosphere
Spatial analysis