Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Jayachandran, Dharanidaran; Smith, Peter; Irfan, Mohammad; Sun, Junhong; Yarborough, John M.; Bomble, Yannick J.; Lam, Eric; Chundawat, Shishir P. S.

doi:10.1002/bit.28484

Title: Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Journal Article · Fri Jun 30 00:00:00 EDT 2023 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.28484· OSTI ID:1987891

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^[1]; Sun, Junhong ^[3]; Yarborough, John M. ^[2];

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Department of Chemical and Biochemical Engineering Rutgers‐The State University of New Jersey Piscataway New Jersey USA
National Renewable Energy Laboratory Biosciences Center Golden Colorado USA
Department of Plant Biology Rutgers‐The State University of New Jersey New Brunswick New Jersey USA

Abstract Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM‐polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM‐based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type‐A CBMs from families 3a and 64 with crystalline cellulose‐I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose‐I using equilibrium binding assays. To compute the adsorption ( nk _on ) and desorption ( k _off ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide‐field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.

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Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC36-08GO28308; SC0019313

OSTI ID:: 1987891

Alternate ID(s):: OSTI ID: 1987895; OSTI ID: 1992017

Report Number(s):: NREL/JA-2700-85128

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Vol. 120 Journal Issue: 8; ISSN 0006-3592

Publisher:: Wiley Blackwell (John Wiley & Sons)Copyright Statement

Country of Publication:: United States

Language:: English

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Title: Engineering and characterization of carbohydrate‐binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

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