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Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

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.

SUBMITTER: Liu L

PROVIDER: S-EPMC3749933 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMechanical strength is a crucial agronomic trait in rice (Oryza sativa), and brittle mutants are thought suitable materials to investigate the mechanism of cell wall formation. So far, almost all brittle mutants are recessive, and most of them are defected in multiple morphologies and/or grain yield, limiting their application in hybrid breeding and in rice straw recycling.ResultsWe identified a semi-dominant brittle mutant Brittle culm19 (Bc19) isolated from the japonica variety Nipponbare through chemical mutagenesis. The mutant showed the same apparent morphologies and grain yield to the wild type plant except for its weak mechanical strength. Its development of secondary cell wall in sclerenchyma cells was affected, along with reduced contents of cellulose, hemicellulose, lignin and sugars in culms and leaves. Positional cloning suggested that the Bc19 gene was allelic to OsCESA4, encoding one of the cellulose synthase A (CESA) catalytic subunits. In this mutant, a C-to-T substitution occurred in the coding sequence of BC19, causing the P507S missense mutation in its encoded product, which was located in the second cytoplasmic region of the OsCESA4 protein. Furthermore, introducing mutant gene Bc19 into the wild-type plant resulted in brittle plants, confirming that the P507S point mutation in OsCESA4 protein was responsible for the semi-dominant brittle phenotype of Bc19 mutant. Reverse correlation was revealed between cellulose contents and expression levels of mutant gene Bc19 among the homozygous mutant, the hybrid F1 plant, and the Bc19 overexpression transgenic plants, implying that gene Bc19 might affect cellulose synthesis in a dosage-dependent manner.ConclusionsBc19, a semi-dominant brittle mutant allele of gene OsCESA4, was identified using map-based cloning approach. The mutated protein of Bc19 possessing the P507S missense mutation behaved in a dosage-dependent semi-dominant manner. Unique brittle effect on phenotype and semi-dominant genetic quality of gene Bc19 indicated its potential application in grain-straw dual-purpose hybrid rice breeding.

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Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

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