Rice Brittle culm 6 encodes a dominant-negative form of CesA protein that perturbs cellulose synthesis in secondary cell walls

The brittle culm (bc) mutants of Gramineae plants having brittle skeletal structures are valuable materials for studying secondary cell walls. In contrast to other recessive bc mutants, rice Bc6 is a semi-dominant bc mutant with easily breakable plant bodies. In this study, the Bc6 gene was cloned by positional cloning. Bc6 encodes a cellulose synthase catalytic subunit, OsCesA9, and has a missense mutation in its highly conserved region. In culms of the Bc6 mutant, the proportion of cellulose was reduced by 38%, while that of hemicellulose was increased by 34%. Introduction of the semi-dominant Bc6 mutant gene into wild-type rice significantly reduced the percentage of cellulose, causing brittle phenotypes. Transmission electron microscopy analysis revealed that Bc6 mutation reduced the cell wall thickness of sclerenchymal cells in culms. In rice expressing a reporter construct, BC6 promoter activity was detected in the culms, nodes, and flowers, and was localized primarily in xylem tissues. This expression pattern was highly similar to that of BC1, which encodes a COBRA-like protein involved in cellulose synthesis in secondary cell walls in rice. These results indicate that BC6 is a secondary cell wall-specific CesA that plays an important role in proper deposition of cellulose in the secondary cell walls.     

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

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).     

BRITTLE CULM1, which encodes a COBRA-like protein,affects the mechanical properties of rice plants

Plant mechanical strength is an important agronomic trait. To understand the molecular mechanism that controls the plant mechanical strength of crops, we characterized the classic rice mutant brittle culm1 (bc1) and isolated BC1 using a map-based cloning approach. BC1, which encodes a COBRA-like protein, is expressed mainly in developing sclerenchyma cells and in vascular bundles of rice. In these types of cells, mutations in BC1 cause not only a reduction in cell wall thickness and cellulose content but also an increase in lignin level, suggesting that BC1, a gene that controls the mechanical strength of monocots, plays an important role in the biosynthesis of the cell walls of mechanical tissues.     

The glycosyltransferase repertoire of the spikemoss Selaginella moellendorffii and a comparative study of its cell wall

Spike mosses are among the most basal vascular plants, and one species, Selaginella moellendorffii, was recently selected for full genome sequencing by the Joint Genome Institute (JGI). Glycosyltransferases (GTs) are involved in many aspects of a plant life, including cell wall biosynthesis, protein glycosylation, primary and secondary metabolism. Here, we present a comparative study of the S. moellendorffii genome across 92 GT families and an additional family (DUF266) likely to include GTs. The study encompasses the moss Physcomitrella patens, a non-vascular land plant, while rice and Arabidopsis represent commelinid and non-commelinid seed plants. Analysis of the subset of GT-families particularly relevant to cell wall polysaccharide biosynthesis was complemented by a detailed analysis of S. moellendorffii cell walls. The S. moellendorffii cell wall contains many of the same components as seed plant cell walls, but appears to differ somewhat in its detailed architecture. The S. moellendorffii genome encodes fewer GTs (287 GTs including DUF266s) than the reference genomes. In a few families, notably GT51 and GT78, S. moellendorffii GTs have no higher plant orthologs, but in most families S. moellendorffii GTs have clear orthologies with Arabidopsis and rice. A gene naming convention of GTs is proposed which takes orthologies and GT-family membership into account. The evolutionary significance of apparently modern and ancient traits in S. moellendorffii is discussed, as is its use as a reference organism for functional annotation of GTs.     

OsCesA4的等位基因Bc7(t)的精细定位和分离

水稻中已经发现并确认了许多脆秆突变体,本研究利用60Co-γ射线诱变粳稻品种中花11得到一脆秆突变体,命名为bc7(t)。与野生型相比,该突变体除了整个植株变脆、纤维素含量降低约10%外,表型与野生型品种相似。遗传分析表明该突变性状受控于单隐性基因,并将该基因精细定位在第1染色体长臂上8.4kb的区段内,基因注释信息表明该区域仅有一个编码纤维素合酶催化亚基(CesA)的基因,与OsCesA4基因等位。进一步的测序分析发现,在突变体中该基因的第10个外显子和第10个内含子的连接处缺失了7个碱基,导致阅读框改变而不能编码功能正常的蛋白。此外,RNA干涉试验表明,将Bc7(t)基因敲除,得到的所有转基因植株表现出类似突变体的脆性。OsCesA4新等位基因的发掘有助于阐明水稻细胞壁的生物合成机理,本文同时也讨论了该突变体用作动物饲料的潜在利用价值。     

Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis

“Brittle culm” mutants found in Gramineae crops are suitable materials to study the mechanism of secondary cell wall formation. Through positional cloning, we have identified a gene responsible for the brittle culm phenotype in rice, brittle culm 3 (bc3). BC3 encodes a member of the classical dynamin protein family, a family known to function widely in membrane dynamics. The bc3 mutation resulted in reductions of 28–36% in cellulose contents in culms, leaves, and roots, while other cell wall components remained unaffected. Reductions of cell wall thickness and birefringence were observed in both fiber (sclerenchyma) and parenchymal cells, together with blurring of the wall’s layered structures. From promoter-GUS analyses, it was suggested that BC3 expression is directly correlated with active secondary cell wall synthesis. These results suggest that BC3 is tightly involved in the synthesis of cellulose and is essential for proper secondary cell wall construction.     

Rice brittleness mutants: a way to open the 'black box' of monocot cell wall biosynthesis

Rice is a model organism for studying the mechanism of cell wall biosynthesis and remolding in Gramineae. Mechanical strength is an important agronomy trait of rice (Oryza sativa L.) plants that affects crop lodging and grain yield. As a prominent physical property of cell walls, mechanical strength reflects upon the structure of different wall polymers and how they interact. Studies on the mechanisms that regulate the mechanical strength therefore consequently results in uncovering the genes functioning in cell wall biosynthesis and remodeling. Our group focuses on the study of isolation of brittle culm (bc) mutants and characterization of their corresponding genes. To date, several bc mutants have been reported. The identified genes have covered several pathways of cell wall biosynthesis, revealing many secrets of monocot cell wall biosynthesis. Here, we review the progress achieved in this research field and also highlight the perspectives in expectancy. All of those lend new insights into mechanisms of cell wall formation and are helpful for harnessing the waste rice straws for biofuel production.     

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

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.     

Isolation of a novel cell wall architecture mutant of rice with defective Arabidopsis COBL4 ortholog BC1 required for regulated deposition of secondary cell wall components

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.     

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.     

Phenotypic characterization, genetic analysis and gene-mapping for a brittle mutant in rice

Plant mechanical strength is an important agronomic trait of rice. An ethyl methane sulfonate （EMS）-induced rice mutant, fragile plant 2 （fp2）, showed morphological changes and reduced mechanical strength. Genetic analysis indicated that the brittle of fp2 was controlled by a recessive gene. The fp2 gene was mapped on chromosome 10. Anatomical analyses showed that the fp2 mutation caused the reduction of cell length and cell wall thickness, increasing of cell width, and the alteration of cell wall structure as well as the vessel elements. The consequence was a global alteration in plant morphology. Chemical analyses indicated that the contents of cellulose and lignin decreased, and hemicelluloses and silicon increased in fp2. These results were different from the other mutants reported in rice. Thus, fp2 might affect the deposition and patterning of microflbrils, the biosynthesis and deposition of cell wall components, which influences the formation of primary and secondary cell walls, the thickness of cell walls, cell elongation and expansion, plant morphology and plant strength in rice.     

The rice dynamin‐related protein DRP2B mediates membrane trafficking,and thereby plays a critical role in secondary cell wall cellulose biosynthesis

Membrane trafficking between the plasma membrane (PM) and intracellular compartments is an important process that regulates the deposition and metabolism of cell wall polysaccharides. Dynamin‐related proteins (DRPs), which function in membrane tubulation and vesiculation are closely associated with cell wall biogenesis. However, the molecular mechanisms by which DRPs participate in cell wall formation are poorly understood. Here, we report the functional characterization of Brittle Culm3 (BC3), a gene encoding OsDRP2B. Consistent with the expression of BC3 in mechanical tissues, the bc3 mutation reduces mechanical strength, which results from decreased cellulose content and altered secondary wall structure. OsDRP2B, one of three members of the DRP2 subfamily in rice (Oryza sativa L.), was identified as an authentic membrane‐associated dynamin via in vitro biochemical analyses. Subcellular localization of fluorescence‐tagged OsDRP2B and several compartment markers in protoplast cells showed that this protein not only lies at the PM and the clathrin‐mediated vesicles, but also is targeted to the trans‐Golgi network (TGN). An FM4‐64 uptake assay in transgenic plants that express green fluorescent protein‐tagged OsDRP2B verified its involvement in an endocytic pathway. BC3 mutation and overexpression altered the abundance of cellulose synthase catalytic subunit 4 (OsCESA4) in the PM and in the endomembrane systems. All of these findings lead us to conclude that OsDRP2B participates in the endocytic pathway, probably as well as in post‐Golgi membrane trafficking. Mutation of OsDRP2B disturbs the membrane trafficking that is essential for normal cellulose biosynthesis of the secondary cell wall, thereby leading to inferior mechanical properties in rice plants.     

A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice

Cellulose synthase (CESA) is a critical catalytic subunit of the cellulose synthase complex responsible for glucan chain elongation. Our knowledge about how CESA functions is still very limited. Here, we report the functional characterization of a rice mutant, brittle culm11, that shows growth retardation and dramatically reduced plant strength. Map-based cloning revealed that all the mutant phenotypes result from a missense mutation in OsCESA4 (G858R), a highly conserved residue at the end of the fifth transmembrane domain. The aberrant secondary cell wall of the mutant plants is attributed to significantly reduced cellulose content, abnormal secondary wall structure of sclerenchyma cells, and overall altered wall composition, as detected by chemical analyses and immunochemical staining. Importantly, we have found that this point mutation decreases the abundance of OsCESA4 in the plasma membrane, probably due to a defect in the process of CESA complex secretion. The data from our biochemical, genetic, and pharmacological analyses indicate that this residue is critical for maintaining the normal level of CESA proteins in the plasma membrane.     

插入含Ds因子的T-DNA产生的水稻脆秆突变株的遗传和分子分析

在构建由农杆菌介导的玉米Ds转座因子插入的水稻转化群体中,得到了一个茎秆等组织发生脆性突变的株系。理化指标定量测定表明,脆性株系的载荷强度和纤维素含量都比正常植株低很多,可溶性糖含量略有减少。对这个突变株的分子检测结果表明Ds因子在脆性株系中为单位点插入。检测了自前3代（T1,T2,T3)植株中T-DNA(Ds)插入与脆性表型的共分离关系。初步结果表明这个突变是T－DNA（Ds)的插入造成的,这个突变基因可能与水稻纤维素合成有关。     

Fucosylated arabinogalactan-proteins are required for full root cell elongation in arabidopsis

The Arabidopsis thaliana mutant mur1 is affected in the biosynthesis of l-fucose and has less than 2% of the normal amounts of this sugar in the cell walls of its aerial parts. Although in roots the reduction of l-fucose is only 40%, this causes a decrease of about 50% in root cell elongation. Since arabinogalactan-proteins (AGPs) are known to play a role in plant cell expansion we studied the composition of mur1 root AGPs. Arabidopsis root AGPs were shown to contain l-fucose, which was reduced in level in mur1 AGPs. In wild-type plants, an l-fucose containing epitope is present in AGPs in the cell wall of differentiating root cells. Addition of eel lectin, which specifically recognizes this epitope, and not fucose in other wall polymers, can phenocopy mur1 roots. Several lines of evidence are presented to support the contention that l-fucose containing root AGPs are required for the full elongation of root cells.     

Golgi-localized UDP-glucose transporter is required for cell wall integrity in rice

Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its broad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity.Key words: UDP-glucose transporter, Golgi apparatus, cell wall polysaccharides, xylan, riceNucleotide sugars mainly generated in cytosol are the substrates for the synthesis of cell wall polysaccharides. Supply of nucleotide sugars is thus a key level for regulation of cell wall components and structure. Mutation in MUR1, an isoform of GDP-D-mannose-4,6-dehydratase, causes reduced amount of GDP-fucose and abnormal xyloglucan structure.^1,2 Disturbance of UDP-rhamnose synthesis via the mutation in RHM2/MUM4 decreases the rhamnogalacturonan I contents in Arabidopsis seeds. Cellulose synthase catalytic subunits (CESAs) generally use cytosolic UDP-glucoses to synthesize cellulose on the plasma membrane. UDP-glucose can be produced either via the catalysis of sucrose by sucrose synthase (SuSy) or through the phosphorylation of glucose-1-phosphate by UDP-glucose pyrophosphorylase (UGPase).³ Suppression of SuSy function in cotton inhibited fiber initiation and elongation.⁴ For the synthesis of noncellulosic polysaccharides occurring inside the Golgi lumen, the cytosolic nucleotide sugars should be translocated inwards by Golgi nucleotide sugar transporters (NSTs).⁵ However, this hypothesis remains to be confirmed, although transport activities have been identified in some plant NSTs.^6–10 Altering the precursor supply may also affect the overall carbon allocation in plants. It is reasonable that substrate regulation often causes pleiotropic effects on cell wall biosynthesis and plant growth. Without genetic resources or mutants on cell wall related NST, the exact evaluation of NSTs'' impacts on cell wall structure and composition is largely delayed. Until recently, we identified a Golgi-localized transporter OsNST1 mutant in rice. This transporter has been found to supply UDP-glucose for the formation of matrix polysaccharides, thereby modulating cellulose biosynthesis.¹¹ Here, we examine these alterations of cell wall polymers at the cellular level. The orientation of cellulose microfibrils and extractability of wall polysaccharides were also compared between the mutant and wild type. All those further our understandings of the functions of NSTs and the synergetic synthesis of different polymers.     

Loss of Cellulose synthase-like F6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles and leaves retain trace amounts of MLG only in specific cell types such as sclerenchyma fibers. These results correlate with the absence of endogenous MLG synthase activity in mutant seedlings and 4-week-old sheaths. Mutant cell walls are weaker in mature stems but not seedlings, and more brittle in both stems and seedlings, compared to wild type. Mutants also display lesion mimic phenotypes in leaves, which correlates with enhanced defense-related gene expression and enhanced disease resistance. Taken together, our results underline a weaker role of MLG in cell expansion than previously thought, and highlight a structural role for MLG in nonexpanding, mature stem tissues in rice.     

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2 (AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1(-)) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3 were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3 antisense plants. In keeping with the mild phenotype compared with Rsw1(-), reductions in total cellulose levels in antisense CesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, like CesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 in rsw1 but were unable to complement that mutant's defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31 degrees C.