Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Lifeng Liu,Mu Zhang,Xiangling Liu,Lanjun Zhang,Keke Shang-Guan,Qian Qian,Yanyun Shi,Yihua Zhou,Jiayang Li,Meixian Yan,Baocai Zhang,Li-Jia Qu

doi:10.1371/journal.pgen.1003704

Abstract

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Highlights

Cellulose, a class of homogenous polymers (b-1,4-glucans), represents the most abundant component of cell walls and play fundamental roles in plant growth and development
Our findings provide evidence that cellulose assembly requires the participation of Brittle Culm1 (BC1), and this is likely to be the case for COB members (COBs) and other COB-like genes (COBLs) proteins as well
By protein gel blotting with this antibody, we demonstrated that BC1 is a membrane protein because it could not be solubilized by high salt (1 M NaCl), alkalinity (0.1 M Na2CO3, pH 11) or a low concentration of detergent (1% Triton X-100)

Summary

Introduction

A class of homogenous polymers (b-1,4-glucans), represents the most abundant component of cell walls and play fundamental roles in plant growth and development. In primary cell walls (PCWs), cellulose microfibrils are cross-linked with pectin, hemicellulose and numerous proteins to define the direction and extent of cell expansion [1]. The disruption of cellulose biosynthesis at this stage generally causes a rapid loss of growth anisotropy [2,3,4,5]. In secondary cell walls (SCWs), cellulose that is embedded in the matrix of hemicellulose and lignin largely determines the mechanical characteristics of the wall [6]. Cellulose deficiency in SCWs often results in collapsed xylem and inferior mechanical strength [7,8,9]. Unraveling the factors that control the quality and quantity of cellulose will facilitate an understanding of plant cell wall biosynthesis and enable us to genetically modify cellulose

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Results

Conclusion