Abstract

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 themore » 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.« less

Authors:

Zegeye, Elias K.  ^[1];

• Search DOE PAGES for author "Zegeye, Elias K."
• Search DOE PAGES for ORCID "0000-0002-5130-582X"

Lin, Vivian S.  ^[2];

• Search DOE PAGES for author "Lin, Vivian S."
• Search DOE PAGES for ORCID "0000-0001-9159-0031"

Nuñez, Jamie R. ^[2]; Sconzo, Nick A. ^[2]; Purvine, Samuel O.  ^[2];

• Search DOE PAGES for author "Purvine, Samuel O."
• Search DOE PAGES for ORCID "0000-0002-2257-2400"

Wright, Aaron T.  ^[3];

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• Search DOE PAGES for ORCID "0000-0002-3172-5253"

Moran, James J.  ^[4]

• Search DOE PAGES for author "Moran, James J."
• Search DOE PAGES for ORCID "0000-0001-9081-9017"

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+ Show Author Affiliations

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)

Publication Date:

Mon Jul 10 00:00:00 EDT 2023

Research Org.:

Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Org.:

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)

OSTI Identifier:

2000746

Report Number(s):

PNNL-SA-182442
Journal ID: ISSN 0038-0717

Grant/Contract Number:  

AC05-76RL01830; DEB 1832042

Resource Type:

Accepted Manuscript

Journal Name:

Soil Biology and Biochemistry

Additional Journal Information:

Journal Volume: 184; Journal ID: ISSN 0038-0717

Publisher:

Elsevier

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; Chitin; Chitinase; Rhizosphere; Spatial analysis; Fluorescence probe