Title: A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Abstract

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminalmore » RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Lastly, our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.« less

Authors:

Purushotham, Pallinti ^[1]; Cho, Sung Hyun ^[2]; Díaz-Moreno, Sara M. ^[3]; Kumar, Manish ^[4]; Nixon, B. Tracy ^[2]; Bulone, Vincent ^[5]; Zimmer, Jochen ^[1]

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+ Show Author Affiliations

1. Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908,
2. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802,
3. Division of Glycoscience, Royal Institute of Technology, Stockholm, SE-10691, Sweden,
4. Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802,
5. Division of Glycoscience, Royal Institute of Technology, Stockholm, SE-10691, Sweden,, Australian Research Council Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, SA, 5064, Australia

Publication Date:

Mon Sep 19 00:00:00 EDT 2016

Research Org.:

Energy Frontier Research Centers (EFRC), Washington, D.C. (United States). Center for Lignocellulose Structure and Formation (CLSF)

Sponsoring Org.:

USDOE Office of Science (SC), Basic Energy Sciences (BES)

OSTI Identifier:

1325834

Alternate Identifier(s):

OSTI ID: 1388837

Grant/Contract Number:  

SC0001090

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 113 Journal Issue: 40; Journal ID: ISSN 0027-8424

Publisher:

Proceedings of the National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; biofuels (including algae and biomass); bio-inspired; membrane; carbon sequestration; materials and chemistry by design; synthesis (self-assembly)