Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex.

B Tracy Nixon,Alison W Roberts,Yaroslava G Yingling,Jung-Goo Lee,Jonathan K Davis,Venu Gopal Vandavasi,Juan Du,Erin Slabaugh,Candace H Haigler,Eric M Roberts,Hugh O’Neill,Katayoun Mansouri,Abhishek Singh

doi:10.1038/srep28696

Abstract

A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In summary, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.

Highlights

Cellulose microfibrils in the land plants and their close algal relatives are synthesized by a six-lobed ‘rosette’ cellulose synthesis complex, or rosette CSC
A persistent question for decades has been: How many cellulose synthase (CESA) are in one rosette CSC, and, how many glucan chains form the fundamental cellulose fibril in plant cell walls? it has long been conjectured that 36 CESAs exist in one rosette CSC9, this idea has been questioned on several grounds
An 18-chain fundamental cellulose microfibril was inferred from the in vitro formation of a trimer from the catalytic domain of CESA1 from Arabidopsis thaliana (AtCESA1; GenBank NP_194967.1), which could represent the cytosolic component of one lobe of the rosette CSC18

Summary

Introduction

Cellulose microfibrils in the land plants and their close algal relatives are synthesized by a six-lobed ‘rosette’ cellulose synthesis complex, or rosette CSC These distinctive multimeric transmembrane protein complexes are revealed by freeze fracture electron microscopy (FF-TEM)[2,3]. This analysis was limited by the use of a generic estimate of TMH area and images of rosette CSCs after shadowing with a thick coating applied unidirectionally from a 45° angle This traditional FF-TEM method resulted in the perception and measurement of the lobes in part through their electron transparent ‘shadows’ where metal was not present, leading to imprecise estimates of lobe shape and dimensions. The data refute the original model of 36 CESAs and support 18 CESAs within the rosette CSC, which is predicted to synthesize an 18-chain fundamental cellulose fibril within cell walls of land plants and their closely related algal progenitors

Methods

Results

Conclusion