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

<div><p>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 <i>BC1</i> and knockdown of <i>BC1</i> expression decrease the crystallite width of cellulose; overexpression of <i>BC1</i> and the CBM-mutated <i>BC1</i>s 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 <i>bc1</i> seedlings with the dyes resulted in insensitive root growth responses in <i>bc1</i> 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.</p></div

BC1 is localized in the cell wall.

<p>(A) Protein blots probed with the indicated antibodies. Antibodies against xyloglucan xyloglucosyl transferase5 (XET5), PIP1s and heat shock protein (HSP) were used for monitoring the cell wall (CW), total membrane (TM) and supernatant (S) protein fractions, respectively. Molecular weights (kD) are indicated at the right. (B–G) Immunogold labeling of BC1 in cells with thin walls (B and C) and thickening cell walls of wild-type (D and E), <i>bc1</i> mutant (F) and <i>BC1RNAi</i> (G) plants. Bars = 0.5 µm in B and C, 5 µm in D and 2 µm in E–G.</p

The CBM and GPI-substitution are essential for the cell wall localization of BC1.

<p>(A) Protein blotting of BC1 in cell wall fractions that were extracted from plants expressing wild-type <i>BC1</i>, <i>BC1</i>^Y46A, <i>BC1</i>^W66A and <i>BC1</i>^W72A. XET5 served as a loading control. (B–E) Immunogold labeling of BC1 in the secondary walls of mature plants expressing wild-type <i>BC1</i> (B), <i>BC1</i>^Y46A (C), <i>BC1</i>^W66A (D) and <i>BC1</i>^W72A (E). (F) Transient expression of GFP-CBM in tobacco leaf epidermal cells. (G) Mannitol-induced plasmolysis to examine GFP-CBM in tobacco epidermal cells. Bars = 1 µm in B–E and 20 µm in F and G.</p

BC1 affects cellulose crystallite size and cellulose content.

<p>Cell wall residues were prepared from the 3^rd internodes from 4-month old wild type, <i>bc1</i>, and indicated transgenic plants.</p>1<p>The cellulose content is determined by Updegraff quantification after TFA hydrolysis and is presented as µg cellulose per mg alcohol-insoluble residues (AIRs), mean value ± SE. Asterisks indicate <i>P</i><0.01 Student's <i>t</i> test with respect to wild type, <i>n</i> = 4.</p>2<p>As determined by Segal method <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003704#pgen.1003704-Segal1" target="_blank">[47]</a> and presented as percentage of crystalline in cell wall components, mean value ± SE. Asterisks indicate <i>P</i><0.01 Student's <i>t</i> test with respect to wild type, <i>n</i> = 3.</p>3<p>As determined by synchrotron X-ray analysis and presented as Å ± SE. Asterisks indicate <i>P</i><0.01 Student's <i>t</i> test with respect to wild type, <i>n</i> = 3.</p>4, 5<p>Expressing the indicated constructs in <i>bc1</i> mutant and wild-type plants, respectively.</p

The CBM in BC1 is a functional domain.

<p>(A) BC1 and BC1 variants used for the transformation assay. (B) RT-PCR analysis, confirming that the transgenic plants with <i>bc1</i> (/<i>bc1</i>) and wild-type (/WT) backgrounds have increased <i>BC1</i> expression levels. (C–J) TEM micrographs of sclerenchyma cell cross-sections from plants overexpressing the wild-type <i>BC1</i> (C and D) and CBM-mutated <i>BC</i>, including <i>BC1</i>^Y46A (E and F), <i>BC1</i>^W66A (G and H), and <i>BC1</i>^W72A (I and J). The dashed rectangles indicate the parts being magnified in the lower panels (D, F, H, and J). Bars = 5 µm (C, E, G, and I) and 1 µm (D, F, H, and J).</p

Biochemical properties of BC1.

<p>(A) SDS-PAGE gels used to separate the total membrane protein from the indicated plants were blotted and probed with anti-BC1 and anti-PIP1s antibodies. A gel stained with Coomassie blue is shown in the right panel. (B) A protein blot probed with anti-BC1 and anti-PIP1s antibodies. The treated proteins were subjected to ultracentrifugation into supernatant (S) and pellet (P) fractions. The total proteins extracted from plants expressing <i>BC3-GFP</i> were probed with anti-GFP antibody, which served as a positive control for the indicated treatments. (C) Protein blotting of buffer (−PLD)- or PLD (+PLD)-treated membrane proteins with anti-BC1 and anti-PIP1s antibodies. The proteins were subjected to ultracentrifugation into supernatant (S) and pellet (P) fractions. (D) Protein blotting of buffer (−PNGase F)- or <i>N</i>-glycosidase (+PNGase F)-treated protein extracts with anti-BC1 and anti-PIP1s antibodies. Molecular weights (kD) are indicated. PIP1s is a plasma membrane aquaporin of rice plants that facilitates the transport of water across the cell membrane, which served as loading or negative controls in these experiments.</p

The CBM of BC1 binds crystalline cellulose.

<p>(A) Relative binding affinity of the CBM for various cell wall substrates. The relative absorbance is derived from the protein blot signals shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003704#pgen.1003704.s003" target="_blank">Figure S3</a>. The data are presented as the mean ± SE (<i>n</i>≥2). (B) Binding affinity of the CBM for crystalline cellulose from plants and commercial products determined via ELISA. The data are presented as the mean ± SE (<i>n</i> = 3). (C) Dissociation constant value for CBM binding to rice crystalline cellulose obtained from a representative experiment. (D) Immunochemical staining of rice crystalline cellulose with recombinant CBM or IIP4 (a negative control) using anti-His and anti-FITC as the primary and secondary antibodies, respectively. Bar = 100 µm. (E) Relative binding affinity of the wild-type and point-mutated CBMs to commercial cellulose (20 µm powder) determined via ELISA. The data are presented as the mean ± SE (<i>n</i>≥2, **<i>P</i><0.01 by Student's <i>t</i>-test).</p

BC1 modulates cellulose crystallization.

<p>(A–D) Immunostaining of CBM with unstained or pre-stained rice crystalline cellulose (0.01% S4B, w/v) using anti-His and anti-FITC as primary and secondary antibodies, respectively. Bar = 100 µm. (E) Effects of S4B on root growth in wild-type and <i>bc1</i> seedlings. Primary root length was measured at the indicated time. MS, Murashige and Skoog medium. The data are presented as the mean ± SE (<i>n</i>≥15). Square brackets indicate the varied responses to one concentration of S4B between wild-type and <i>bc1</i> seedlings.</p