Abstract
In plants, changes in cell size and shape during development fundamentally depend on the ability to synthesize and modify cell wall polysaccharides. The main classes of cell wall polysaccharides produced by terrestrial plants are cellulose, hemicelluloses, and pectins. Members of the cellulose synthase (CESA) and cellulose synthase-like (CSL) families encode glycosyltransferases that synthesize the β-1,4-linked glycan backbones of cellulose and most hemicellulosic polysaccharides that comprise plant cell walls. Cellulose microfibrils are the major load-bearing component in plant cell walls and are assembled from individual β-1,4-glucan polymers synthesized by CESA proteins that are organized into multimeric complexes called CESA complexes, in the plant plasma membrane. During distinct modes of polarized cell wall deposition, such as in the tip growth that occurs during the formation of root hairs and pollen tubes or de novo formation of cell plates during plant cytokinesis, newly synthesized cell wall polysaccharides are deposited in a restricted region of the cell. These processes require the activity of members of the CESA-like D subfamily. However, while these CSLD polysaccharide synthases are essential, the nature of the polysaccharides they synthesize has remained elusive. Here, we use a combination of genetic rescue experiments with CSLD-CESA chimeric proteins, in vitro biochemical reconstitution, and supporting computational modeling and simulation, to demonstrate that Arabidopsis (Arabidopsis thaliana) CSLD3 is a UDP-glucose-dependent β-1,4-glucan synthase that forms protein complexes displaying similar ultrastructural features to those formed by CESA6.
Highlights
Cellulose is one of the most abundant organic polymers in nature and is the principal component of the plant cell wall, providing most of its tensile strength (Baskin, 2005; Cosgrove, 2005)
Cellulose Synthase (CESA) and CSLD proteins share overall membrane topology and maintain high degrees of sequence identity, especially in the central domain where critical catalytic residues are absolutely conserved (Figure 1A; (Morgan et al, 2013; Sethaphong et al, 2013; Slabaugh et al, 2014)). We previously used this structural similarity to demonstrate that a fluorescently-tagged chimeric fusion protein in which the CSLD3 catalytic region was replaced with the corresponding CESA6 catalytic domain was able to quantitatively rescue kjk-2 root hair defects (Park et al, 2011). While these results indicated that a chimeric CSLD3 fusion could restore root hair growth, it remained unclear whether CSLD catalytic domains could replace CESA sequences
These results indicate that Citrine-CESA6:D3CD chimera proteins are capable of integrating into primary cell wall cellulose synthase complexes (CSCs), and based on their similar speeds, display similar cell wall biosynthesis characteristics
Summary
Cellulose is one of the most abundant organic polymers in nature and is the principal component of the plant cell wall, providing most of its tensile strength (Baskin, 2005; Cosgrove, 2005). Cellulose microfibrils contain multiple β-1,4-glucan chains that associate via intermolecular hydrogen bonds, and are synthesized by large, membrane-localized complexes called “rosette complexes” (Baskin, 2005; Cosgrove, 2005). In Arabidopsis thaliana, CESA proteins interact to form rosette subunits, and six of these subunits assemble into multimeric rosette complexes, often referred to as cellulose synthase complexes (CSCs) (Kimura et al, 1999). In vitro reconstitution of cellulose synthase activity was observed in proteoliposomes containing only Populus tremula x tremuloides CESA8 (PttCESA8) or Physcomitrella patens CESA8 (PpCESA8) (Purushotham et al, 2016; Cho et al, 2017), highlighting that our understanding of the composition of functional cellulose synthase rosette complexes remains incomplete
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