Brittle culm15 encodes a membrane-associated chitinase-like protein required for cellulose biosynthesis in rice

Abstract

Plant chitinases, a class of glycosyl hydrolases, participate in various aspects of normal plant growth and development, including cell wall metabolism and disease resistance. The rice (Oryza sativa) genome encodes 37 putative chitinases and chitinase-like proteins. However, none of them has been characterized at the genetic level. In this study, we report the isolation of a brittle culm mutant, bc15, and the map-based cloning of the BC15/OsCTL1 (for chitinase-like1) gene affected in the mutant. The gene encodes the rice chitinase-like protein BC15/OsCTL1. Mutation of BC15/OsCTL1 causes reduced cellulose content and mechanical strength without obvious alterations in plant growth. Bioinformatic analyses indicated that BC15/OsCTL1 is a class II chitinase-like protein that is devoid of both an amino-terminal cysteine-rich domain and the chitinase activity motif H-E-T-T but possesses an amino-terminal transmembrane domain. Biochemical assays demonstrated that BC15/OsCTL1 is a Golgi-localized type II membrane protein that lacks classical chitinase activity. Quantitative real-time polymerase chain reaction and β-glucuronidase activity analyses indicated that BC15/OsCTL1 is ubiquitously expressed. Investigation of the global expression profile of wild-type and bc15 plants, using Illumina RNA sequencing, further suggested a possible mechanism by which BC15/OsCTL1 mediates cellulose biosynthesis and cell wall remodeling. Our findings provide genetic evidence of a role for plant chitinases in cellulose biosynthesis in rice, which appears to differ from their roles as revealed by analysis of Arabidopsis (Arabidopsis thaliana).

Figure 1.

Comparison of the mechanical strength and secondary cell wall structure between bc15 and wild-type (WT) plants. A and B, The mutant internodes (A) and leaves (B) are easily broken. C and D, Triple measurements of the break force of internodes (C) and leaves (D). E to H, Transmission electron micrographs of the sclerenchyma cell walls of the wild type (E) and bc15 (G). F and H are enlargements of the boxed areas in E and G, respectively. Bars = 5 μm (E and G) and 1 μm in (F and H). [See online article for color version of this figure.]

Figure 2.

Map-based cloning of the BC15 gene. A, The mutation locus was mapped to a 114-kb region between markers RM242 and RM160 on chromosome 9. BC15 is the gene of Os09g32080. The point mutation changes Ala at position 213 to Leu in bc15. B, Constructs for complementation and biochemical assays. C, Three-month-old wild-type plant, bc15 mutant plant, and transgenic plant expressing pBC15F. Bar = 15 cm. D, Digestion of the amplified DNA fragments that cover the mutation site with BstUI to confirm the genotypes of wild-type (WT), bc15, and transgenic rice plants. M, Marker.

Figure 3.

BC15/OsCTL1 is an N-glycosylated membrane protein. A, Prediction of the domain structure of BC15/OsCTL1. TM, Transmembrane domain. B, Protein gel blotting of BC15-GFP in the fractionated total proteins with anti-GFP antibody. The marker protein antibodies that recognize CESA9, HSP, and XET5 were used for monitoring the success of fractionation. S, Supernatant; TM, total membrane; CW1, cell wall fraction extracting with CaCl₂; CW2, cell wall fraction extracting with LiCl. C, Extraction of the total membrane fraction of transgenic plants expressing BC15-GFP with different chemicals. Each treatment was separated into supernatant (S) and microsomes (M) and probed with anti-GFP and OsCESA9 antibodies. D, Protein gel blotting BC15-GFP in the absence (−) and presence (+) of PNGase F.

Figure 4.

BC15/OsCTL1 is a Golgi-localized protein. A, Protein gel blotting BC15/OsCTL1-GFP in fractionated microsomes (M), indicating that BC15/OsCTL1 is mainly present in the endomembrane system. PEG represents the plasma membrane fraction, and DEX represents the endomembrane fraction. Arf and PIP1s are two marker proteins predominantly present in the endomembrane and plasma membrane fractions, respectively. Fractionation of the microsomes extracted from BC15/OsCTL1-GFP transgenic plants is described in “Materials and Methods.” B, Root epidermal cells of transgenic plants expressing pBC15GFP, showing dot-like GFP signals in the intracellular compartments. DIC, Differential interference contrast. Bar = 50 µm. C, A rice protoplast cell coexpressing BC15/OsCTL1-GFP, a Golgi marker (Man49-RFP), and a merged image, indicating that BC15/OsCTL1 is Golgi localized. Bar = 5 µm. D, Microsomal fractions of transgenic plants expressing pBC15GFP were incubated with (+) or without (−) proteinase K in the presence (+) or absence (−) of Triton X-100. LD, Loading control, comprising a Coomassie blue-stained gel.

Figure 5.

Expression pattern of BC15/OsCTL1. A, qRT-PCR analysis of BC15 expression in various rice organs, using the Ubiquitin5 (UBQ5) gene as an internal control. B to G, GUS activity staining assay in various organs of BC15pro::GUS transgenic plants, showing GUS signals in a rice seedling (B), including the elongation root regions (C), the vascular bundles of root mature regions (D), and the leaf lamina joint (E). GUS activity was also observed in mature plants, such as in flower branches and glumes (F) and vascular bundles of mature internodes (G). Arrows show the GUS activity signal. Bars = 1.0 cm (B, E, and F) and 100 µm (C, D, and G).

Figure 6.

Enzyme activity assay. Expression of OsCTL1 and empty vector is shown in the wild type (A and B) and a chitinase mutant strain (C and D), indicating that OsCTL1 could not rescue the mutant yeast to the aggregation state as observed in the wild-type strain (BY4741).

Figure 7.

RNAseq analysis of the genes expressed in wild-type and bc15 internodes. A, Statistical analysis of reads from the wild type (WT) and bc15, showing the high quality of RNAseq data. B, Number of genes that are up-regulated and down-regulated in bc15 compared with those in the wild type. C, Scatterplot to show the overall expression alterations in bc15. Red and green spots indicate the up-regulated and down-regulated genes in bc15, respectively.