Elucidating the Mechanisms of Assembly and Subunit Interaction of the Cellulose Synthase Complex of Arabidopsis Secondary Cell Walls

Abstract

Cellulose is the most abundant biopolymer in nature; however, questions relating to the biochemistry of its synthesis including the structure of the cellulose synthase complex (CSC) can only be answered by the purification of a fully functional complex. Despite its importance, this goal remains elusive. The work described here utilizes epitope tagging of cellulose synthase A (CESA) proteins that are known components of the CSC. To avoid problems associated with preferential purification of CESA monomers, we developed a strategy based on dual epitope tagging of the CESA7 protein to select for CESA multimers. With this approach, we used a two-step purification that preferentially selected for larger CESA oligomers. These preparations consisted solely of the three known secondary cell wall CESA proteins CESA4, CESA7, and CESA8. No additional CESA isoforms or other proteins were identified. The data are consistent with a model in which CESA protein homodimerization occurs prior to formation of larger CESA oligomers. This suggests that the three different CESA proteins undergo dimerization independently, but the presence of all three subunits is required for higher order oligomerization. Analysis of purified CESA complex and crude extracts suggests that disulfide bonds and noncovalent interactions contribute to the stability of the CESA subunit interactions. These results demonstrate that this approach will provide an excellent framework for future detailed analysis of the CSC.Cellulose, a polymer of ␤(1-4)-linked glucose, is the major component of most plant cell walls. In higher plants cellulose is synthesized at the plasma membrane by a very large complex that is believed to simultaneously synthesize many individual glucose chains that hydrogen bond together to form a microfibril (1-4). The cellulose synthase complex (CSC) 2 has been visualized in freeze fracture studies as a rosette structure resembling a hexamer with six distinct lobes (5-7). Most estimates suggest that each lobe of the rosette is itself a hexamer, such that the CSC is a 36-mer (4,8,9). Although the CSC is likely to be a major factor in determining microfibril structure, very little information is known about its organization. The only components of the CSC that have been identified to date are the cellulose synthase A (CESA) proteins that are presumed to be the catalytic subunits of the CSC (1, 4, 10 -12).The CESA family has been most intensively studied in Arabidopsis, and these studies have utilized the characterization of a wide range of mutants (4, 11). In primary cell walls a number of CESA proteins have been shown to be required for cellulose synthesis. Both CESA1 and CESA3 are essential (13-17). In contrast, CESA2, CESA5, and CESA9 appear to be partially redundant with CESA6 (18, 19). The situation is different in the secondary cell walls of developing xylem vessels where three nonredundant CESA proteins are required for cellulose synthesis: CESA4 (20), CESA7 (21), and CESA8 (22). Mutations compromising the function of any one of these t...

Tools for Cellulose Analysis in Plant Cell Walls

“…This opens a great opportunity to perform detailed biochemical analysis of the cellulose synthase complex when a purified preparation can be obtained. To date, this has been extremely challenging, but important progress has been made recently in this area with the purification of complexes from Arabidopsis using dual-epitope tagging and the specific corresponding purification steps (Atanassov et al, 2009). However, it was not possible in this work to search for enzymatic activity, because the purification procedure was not efficient enough.…”

Two Complementary Mechanisms Underpin Cell Wall Patterning during Xylem Vessel Development

“…Cellulose is synthesized at the plasma membrane by large cellulose synthase (CesA) complexes (CSCs; Schneider et al, 2016). The CSCs are composed of a heterotrimeric configuration of 18 to 24 CesAs where CesA1,CesA3,CesA2,5,6, and 9) CesAs produce primary wall cellulose in Arabidopsis thaliana, and CesA4, CesA7, and CesA8 comprise the CSCs necessary to make secondary wall cellulose (Persson et al, 2007;Desprez et al, 2007;Taylor et al, 2003;Atanassov et al, 2009).…”

Complexes with Mixed Primary and Secondary Cellulose Synthases Are Functional in Arabidopsis Plants

“…The interaction between the different CESA proteins in the primary and secondary rosettes has been characterized previously by coimmunoprecipitation and yeast two-hybrid methods, showing interaction patterns with similarities between primary and secondary CESAs (Taylor et al, 2000;Desprez et al, 2007;Wang et al, 2008;Atanassov et al, 2009;Timmers et al, 2009). These results suggest that despite the ancient divergence of the families, the complexes may have retained the same positioning of the CESAs in the complex with respect to each other.…”