Engineering and Characterization of Carbohydrate-Binding Modules for Imaging Cellulose Fibrils Biosynthesis in Plant Protoplasts

Dharanidaran Jayachandran, Peter Smith, Mohammed Irfan, Junhong Sun, John Yarbrough, Yannick Bomble, Eric Lam, Shishir Chundawat

Research output: Contribution to journalArticlepeer-review

2 Scopus Citations

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 (nkon) and desorption (koff) 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.

Original languageAmerican English
Pages (from-to)2253-2268
Number of pages16
JournalBiotechnology and Bioengineering
Volume120
Issue number8
DOIs
StatePublished - 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.

NREL Publication Number

  • NREL/JA-2700-85128

Keywords

  • Arabidopsis plant protoplasts
  • carbohydrate-binding module
  • cell wall biosynthesis
  • cellulose microfibrils
  • live-cell imaging
  • quartz crystal microbalance with dissipation

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