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.     

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.     

Characterization and gene mapping of a brittle culm mutant of diploid wheat (Triticum monococcum L.) with irregular xylem vessels development

Diploid wheat, Triticum monococcum L. (einkorn) is an ideal plant material for wheat functional genomics. Brittle culm mutant was identified by screening of the ethyl methane sulphonate-treated M ₂ progenies of a T. monococcum accession pau14087 by banding the plant parts manually. The brittle culm mutant with drooping leaves, early flowering, reduced tiller numbers and susceptible to lodging had also exhibited brittleness in all plant parts than the wild-type parents. Comprehensive mechanical strength, histological, biochemical, scanning electron microscopy, and Fourier transform infrared spectroscopy analyses of brittle culm mutant supplemented and complemented the findings that the mutant had defective cellulose biosynthesis pathway and deposition of cell wall materials on secondary cell wall of mechanical tissues. Microscopic studies demonstrated that the decrease in cellulose contents resulted in the irregular cell wall organization in xylem vessels of leaf vascular bundles. To map the brc5 mutant, mapping populations were developed by crossing the brittle culm mutant with wild Triticum boeoticum acc. pau5088, having non-brittle characters. The brittle culm mutation was mapped between SSR markers, Xcfd39 and Xgwm126 on 5A^mL chromosome of T. monococcum, with genetic distances of 2.6 and 4.8 cM, respectively. The brc5 mutant mapped on 5A^mL, being distinct from a previously mapped brittle culm mutant in wheat, has been designated as brc5. The work on fine mapping and map-based cloning of brc5 gene regulating synthesis and deposition of cellulose on the secondary cell wall is in progress.     

BC10, a DUF266-containing and Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.)

Glycosyltransferases (GTs) are one of the largest enzyme groups required for the synthesis of complex wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function(s) of most GTs in cell-wall biosynthesis and assembly have remained elusive. We report here the isolation and in-depth characterization of a brittle rice mutant, brittle culm 10 ( bc10 ). bc10 plants show pleiotropic phenotypes, including brittleness of the plant body and retarded growth. The BC10 gene was cloned through a map-based approach, and encodes a Golgi-located type II membrane protein that contains a domain designated as 'domain of unknown function 266' (DUF266) and represents a multiple gene family in rice. BC10 has low sequence similarity with the domain to a core 2 β-1,6- N- acetylglucosaminyltransferase (C2GnT), and its in vitro enzymatic activity suggests that it functions as a glycosyltransferase. Monosaccharide analysis of total and fractioned wall residues revealed that bc10 showed impaired cellulose biosynthesis. Immunolocalization and isolation of arabinogalactan proteins (AGPs) in the wild-type and bc10 showed that the level of AGPs in the mutant is significantly affected. BC10 is mainly expressed in the developing sclerenchyma and vascular bundle cells, and its deficiency causes a reduction in the levels of cellulose and AGPs, leading to inferior mechanical properties.     

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.     

Culm strength of barley : correlation among maximum bending stress, cell wall dimensions, and cellulose content

Grass culms are known to differ in breaking strength, but there is little physicochemical data to explain the response. The fourth internode of four brittle and two nonbrittle barley (Hordeum vulgare L.) strains were used for physical and chemical studies of culm strength. Inner and outer culm diameters of brittle strains (3.6 ± 0.2 and 5.0 ± 0.1 millimeters) were not significantly different from those of nonbrittle strains (3.9 ± 0.2 and 5.2 ± 0.2 millimeters). Maximum bending stress, at which the culm was broken, was 192 ± 34 g/mm² for brittle and 490 ± 38 g/mm² for nonbrittle strains. Wall thickness and cell dimensions of epidermal, sclerenchyma, and parenchyma cells were measured in culm cross sections. The area of cell wall per unit cell area for each tissue was significantly correlated with the maximum bending stress (r = 0.93 for epidermis, 0.90 for sclerenchyma, and 0.84 for parenchyma). Cell walls of brittle culms had 6 to 64% as much cellulose content as those of nonbrittle culms. Maximum bending stress correlated significantly with cellulose content of the cell walls (r = 0.93), but not with the contents of noncellulosic compounds. The lower cellulose content of the brittle culm was significantly correlated with brittleness.     

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.     

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.     

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).     

Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant

Key message

Overexpression of cotton cellulose synthase like D3 (GhCSLD3) gene partially rescued growth defect of atcesa6 mutant with restored cell elongation and cell wall integrity mainly by enhancing primary cellulose production.

Abstract

Among cellulose synthase like (CSL) family proteins, CSLDs share the highest sequence similarity to cellulose synthase (CESA) proteins. Although CSLD proteins have been implicated to participate in the synthesis of carbohydrate-based polymers (cellulose, pectins and hemicelluloses), and therefore plant cell wall formation, the exact biochemical function of CSLD proteins remains controversial and the function of the remaining CSLD genes in other species have not been determined. In this study, we attempted to illustrate the function of CSLD proteins by overexpressing Arabidopsis AtCSLD2, -3, -5 and cotton GhCSLD3 genes in the atcesa6 mutant, which has a background that is defective for primary cell wall cellulose synthesis in Arabidopsis. We found that GhCSLD3 overexpression partially rescued the growth defect of the atcesa6 mutant during early vegetative growth. Despite the atceas6 mutant having significantly reduced cellulose contents, the defected cell walls and lower dry mass, GhCSLD3 overexpression largely restored cell wall integrity (CWI) and improved the biomass yield. Our result suggests that overexpression of the GhCSLD protein enhances primary cell wall synthesis and compensates for the loss of CESAs, which is required for cellulose production, therefore rescuing defects in cell elongation and CWI.

     

Three AtCesA6‐like members enhance biomass production by distinctively promoting cell growth in Arabidopsis

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6‐like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild‐type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6‐like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.     

Factors affecting the yield and properties of bacterial cellulose

Acetobacter xylinum E₂₅ has been applied in our studies in order to find optimal culture conditions for effective bacterial cellulose (BC) production. The strain displays significantly higher stability in BC production under stationary culture conditions. In contrast, intensive agitation and aeration appear to drastically reduce cellulose synthesis since such conditions induced formation of spontaneous cellulose nonproducing mutants (Cel−), which dominated in the culture. Mutation frequency strictly depends on the medium composition in agitated cultures. Enrichment of the standard SH and Yamanaka media with 1% ethanol significantly enhanced BC production in stationary cultures. Horizontal fermentors equipped with rotating discs or rollers were successfully applied in order to improve culture conditions. Relatively slow rotation velocity (4 rpm) and large surface area enabling effective cell attachment are optimal parameters for cellulose production. Physical properties of BC samples synthesized either in stationary cultures or in a horizontal fermentor revealed that cellulose from stationary cultures demonstrated a much higher value of Young's modulus, but a much lower value of water-holding capacity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189–195 doi:10.1038/sj.jim.7000303 Received 01 March 2002/ Accepted in revised form 18 July 2002     

OsBC1L4 encodes a COBRA-like protein that affects cellulose synthesis in rice

Plant morphogenesis is highly dependent on the regulation of cell division and expansion. The organization of the cellulose microfibrils in the cell wall is a key determinant of cell expansion. Previously, a dwarf mutant with fewer tillers, Osbc1l4 (Oryza sativa brittle culm 1 like 4), was identified by screening a rice T-DNA insertion mutant library. It is reported here that OsBC1L4 encodes a COBRA-like protein that exhibits typical structural features of a glycosylphosphatidylinositol-anchor protein. The T-DNA insertion in OsBC1L4 results in abnormal cell expansion. A decrease in cellulose content but the increase in pectin and starch contents was identified in Osbc1l4 mutants by measuring the content of wall components. OsBC1L4 was expressed in all tissues/organs examined, with a low level in leaves. OsBC1L4 protein is mainly located in the cell wall and plasma membrane. Correlation analysis indicated that the expression of OsBC1L4 was highly correlated to that of several primary wall-forming cellulose synthase genes (CESAs). Moreover, the expression level of several cellulose-related genes is increased in Osbc1l4 mutants, which suggests that a feedback mechanism may exist during cellulose synthesis.     

Deposition of glycine-rich structural protein in xylem cell walls of french bean seedlings is independent of lignification

Lignin biosynthesis was inhibited in young bean seedlings by 2-aminoindan-2-phosphonic acid (AIP). AIP is a specific and potent inhibitor of phenylalanine ammonialyase, an enzyme involved in lignin biosynthesis. At a concentration of 100 μM AIP in the growth medium, no lignin could be detected in roots and hypocotyls of 7- or 9-day-old seedlings when stained with phloroglucinol/HCl. At an AIP concentration of 70 μM only a very weak lignification was observed, whereas at 30 μM, no inhibition of lignification was detectable. Glycine-rich protein GRP 1.8, a cell wall protein present in protoxylem of beans, was studied by immunocytochemistry in hypocotyls grown in the presence of 100 μM AIP. No difference of the GRP deposition pattern at sites of normally lignified secondary cell wall thickenings, as well as along the protoxylem vessels, was found in unlignified tissue when compared to controls. The cell-type specific synthesis of GRP 1.8 was not affected by AIP. Thus, deposition of the GRP 1.8 structural cell wall protein is independent of lignification, and lignin does not act as an essential scaffold for correct GRP 1.8 deposition in the complex wall structure of xylem.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

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.     

Root hair-specific disruption of cellulose and xyloglucan in <Emphasis Type="Italic">AtCSLD3</Emphasis> mutants,and factors affecting the post-rupture resumption of mutant root hair growth

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5oC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy