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。     

Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall

The physicochemical nature of the cell wall was determined in the fourth internode of three isogenic brittle mutants of barley (Hordeum vulgare L.) and corresponding nonbrittle strains. Cellulose contents of the brittle culms were 17.5 to 20.3% of those of corresponding nonbrittle strains. No major difference was found in lignin and noncellulose components (except glucose) between brittle and nonbrittle strains. Maximum bending stresses of brittle culms were 38.0 to 54.2% of those of corresponding nonbrittle strains. The degree of polymerization of cellulose, measured by viscometry, was similar between the brittle and the nonbrittle strains. Mole number of cellulose molecules in a unit length of brittle culms, calculated by dividing cellulose mass by molecular weight, was 7.7 to 17.3% of those of the nonbrittle strains. These results indicate that brittleness of mutant culms is due to fewer numbers of cellulose molecules in the cell walls.     

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。     

Increase in the Amount of celA1 Protein in Tobacco BY-2 Cells by a Cellulose Biosynthesis Inhibitor, 2,6-dichlorobenzonitrile

The biochemical analysis of cellulose biosynthesis by plantshas been a difficult problem due to the lack of a reliable assayprocedure for cellulose synthase activity. Recently, the celAlgene was cloned from cotton fiber, and this gene was identifiedfrom the rsw1 mutant of Arabidopsis as a catalytic subunit ofcellulose synthase (Arioli et al. 1998). The cloning of thesegenes enables us to obtain specific antibodies against cellulosesynthase. A highly specific antibody against celAl protein wasprepared and used to detect the protein from microsomal fractionof tobacco BY-2 cells. The quantity of celAl protein in microsomalfraction of normal BY-2 cells was under the detection limit,although they contained a large quantity of cellulose. In contrast,cells habituated to 1 µM DCB (a specific inhibitor ofcellulose biosynthesis) produced 1/10 of cellulose content ofthe normal cells, but had much more celAl protein than the normalcells. The amount of polysaccharides in the EDTA-soluble fractionwas relatively increased in habituated cells. The results suggestthat celAl protein is stabilized upon DCB binding and that thecrystallization of cellulose microfibrils is inhibited simultaneously. (Received January 28, 1998; Accepted May 7, 1998)     

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.     

水稻脆杆突变体bcm581-1茎杆形态结构观察、理化测定和遗传分析

在根癌农杆菌介导的Ds转座因子转化的水稻株系中,发现了脆杆突变体bcm581-1.经Basta除草剂抗性检测和PCR检测,这个脆杆突变不是由Ds转座因子插入引起;通过光学显微镜观察发现突变体的小维管束数目多于对照,小维管束之间的凹陷比对照深,而皮层纤维细胞层数少于对照;扫描电镜观察发现突变体表皮细胞外侧的硅质没有对照丰富.虽然,单位面积内的细胞数与对照相近.但是,突变体的细胞壁薄,维管束内纤维细胞壁的加厚程度也低于对照.因此,细胞腔明显比对照大.茎杆的力学测定结果表明突变体的载荷低于对照9.6倍;延伸低5.4倍;延伸率低6.9倍;应力低6倍.茎杆的相对含水量和粗纤维含量测定表明突变体的含水量高于对照3.5%,粗纤维含量则低于对照8.12%.bcm581-1与中花11号杂交试验显示,F1植株全部正常,F2群体中,正常杆和脆杆以31分离,以中花11号为回交亲本的F1B1植株,表现正常;而以脆杆为回交亲本的F1B1植株,正常茎杆和脆杆则以11分离,结果表明bcm581-1的茎杆变脆是受隐性单基因控制的突变.     

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.     

水稻脆杆突变体bcm581-1茎杆形态结构观察、理化测定和遗传分析

在根癌农杆菌介导的Ds转座因子转化的水稻株系中,发现了脆杆突变体bcm581-1。经Basta除草剂抗性检测和PCR检测,这个脆杆突变不是由Ds转座因子插入引起;通过光学显微镜观察发现突变体的小维管束数目多于对照,小维管束之间的凹陷比对照深,而皮层纤维细胞层数少于对照;扫描电镜观察发现突变体表皮细胞外侧的硅质没有对照丰富。虽然,单位面积内的细胞数与对照相近。但是,突变体的细胞壁薄,维管束内纤维细胞壁的加厚程度也低于对照。因此,细胞腔明显比对照大。茎杆的力学测定结果表明：突变体的载荷低于对照9．6倍;延伸低5．4倍;延伸率低6．9倍;应力低6倍。茎杆的相对含水量和粗纤维含量测定表明：突变体的含水量高于对照3．5％,粗纤维含量则低于对照8．12％。bcm581-1与中花11号杂交试验显示,F1植株全部正常,F2群体中,正常杆和脆杆以3：1分离,以中花11号为回交亲本的F181植株,表现正常;而以脆杆为回交亲本的F181植株,正常茎杆和脆杆则以1：1分离,结果表明：bcm581-1的茎杆变脆是受隐性单基因控制的突变。     

Development of cellulose synthesizing complexes inBoergesenia andValonia

Summary The development of linear cellulose synthesizing complexes (=TCs) of two selected siphonocladalean algae,Boergesenia forbesii andValonia ventricosa was investigated by following the time course of the regeneration of cell walls with the freeze fracture technique after aplanospore induction. The following structural changes of TC development were examined: (1) TCs initiatede novo; (2) the first nucleation of TC subunits occurs within 2 hr inBoergesenia and 5 hr inValonia after aplanospore induction, immediately followed by the assembly of cellulose microfibrils; (3) TCs increase their length during the assembly of randomly oriented microfibrils; and, (4) TCs stop increasing in length after the assembly of ordered microfibrils begins, with some time lag. The data demonstrate that linear TCs are not artificial products but dynamic entities which are involved in the assembly of cellulose microfibrils.     

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.     

Maize Brittle stalk2 encodes a COBRA-like protein expressed in early organ development but required for tissue flexibility at maturity

The maize (Zea mays) brittle stalk2 (bk2) is a recessive mutant, the aerial parts of which are easily broken. The bk2 phenotype is developmentally regulated and appears 4 weeks after planting, at about the fifth-leaf stage. Before this time, mutants are indistinguishable from wild-type siblings. Afterward, all organs of the bk2 mutants turn brittle, even the preexisting ones, and they remain brittle throughout the life of the plant. Leaf tension assays and bend tests of the internodes show that the brittle phenotype does not result from loss of tensile strength but from loss in flexibility that causes the tissues to snap instead of bend. The Bk2 gene was cloned by a combination of transposon tagging and a candidate gene approach and found to encode a COBRA-like protein similar to rice (Oryza sativa) BC1 and Arabidopsis (Arabidopsis thaliana) COBRA-LIKE4. The outer periphery of the stalk has fewer vascular bundles, and the sclerids underlying the epidermis possess thinner secondary walls. Relative cellulose content is not strictly correlated with the brittle phenotype. Cellulose content in mature zones of bk2 mature stems is lowered by 40% but is about the same as wild type in developing stems. Although relative cellulose content is lowered in leaves after the onset of the brittle phenotype, total wall mass as a proportion of dry mass is either unchanged or slightly increased, indicating a compensatory increase in noncellulosic carbohydrate mass. Fourier transform infrared spectra indicated an increase in phenolic ester content in the walls of bk2 leaves and stems. Total content of lignin is unaffected in bk2 juvenile leaves before or after appearance of the brittle phenotype, but bk2 mature and developing stems are markedly enriched in lignin compared to wild-type stems. Despite increased lignin in bk2 stems, loss of staining with phloroglucinol and ultraviolet autofluorescence is observed in vascular bundles and sclerid layers. Consistent with the infrared analyses, levels of saponifiable hydroxycinnamates are elevated in bk2 leaves and stems. As Bk2 is highly expressed during early development, well before the onset of the brittle phenotype, we propose that Bk2 functions in a patterning of lignin-cellulosic interactions that maintain organ flexibility rather than having a direct role in cellulose biosynthesis.     

TIP CELL GROWTH AND THE FREQUENCY AND DISTRIBUTION OF CELLULOSE MICROFIBRIL-SYNTHESIZING COMPLEXES IN THE PLASMA MEMBRANE OF APICAL SHOOT CELLS OF THE RED ALGA PORPHYRA YEZOENSIS1

The supramolecular organization of the plasma membrane of apical cells in shoot filaments of the marine red alga Porphyra yezoensis Ueda (conchocelis stage) was studied in replicas of rapidly frozen and fractured cells. The protoplasmic fracture (PF) face of the plasma membrane exhibited both randomly distributed single particles (with a mean diameter of 9.2 ± 0.2 nm) and distinct linear cellulose microfibril-synthesizing terminal complexes (TCs) consisting of two or three rows of linearly arranged particles (average diameter of TC particles 9.4 plusmn; 0.3 nm). The density of the single particles of the PF face of the plasma membrane was 3000 μm^?2, whereas that of the exoplasmic fracture face was 325 μm^?2. TCs were observed only on the PF face. The highest density of TCs was at the apex of the cell (mean density 23.0 plusmn; 7.4 TCs μm^?2 within 5 μm from the tip) and decreased rapidly from the apex to the more basal regions of the cell, dropping to near zero at 20 μm. The number of particle subunits of TCs per μm² of the plasma membrane also decreased from the tip to the basal regions following the same gradient as that of the TC density. The length of TCs increased gradually from the tip (mean length 46.0 plusmn; 1.4 nm in the area at 0–5 μm from the tip) to the cell base (mean length 60.0 plusmn; 7.0 μm in the area at 15–20 μm). In the very tip region (0–4 μm from the apex), randomly distributed TCs but no microfibril imprints were observed, while in the region 4–9 μm from the tip microfibril imprints and TCs, both randomly distributed, occurred. Many TCs involved in the synthesis of cellulose microfibrils were associated with the ends of microfibril imprints. Our results indicate that TCs are involved in the biosynthesis, assembly, and orientation of cellulose microfibrils and that the frequency and distribution of TCs reflect tip growth (polar growth) in the apical shoot cell of Porphyra yezoensis. Polar distribution of linear TCs as “cellulose synthase” complexes within the plasma membrane of a tip cell was recorded for the first time in plants.     

THE SITES OF CELLULOSE SYNTHESIS IN ALGAE: DIVERSITY AND EVOLUTION OF CELLULOSE-SYNTHESIZING ENZYME COMPLEXES

Information on the sites of cellulose synthesis and the diversity and evolution of cellulose-synthesizing enzyme complexes (terminal complexes) in algae is reviewed. There is now ample evidence that cellulose synthesis occurs at the plasma membrane-bound cellulose synthase, with the exception of some algae that produce cellulosic scales in the Golgi apparatus. Freeze-fracture studies of the supramolecular organization of the plasma membrane support the view that the rosettes (a six-subunit complex) in higher plants and both the rosettes and the linear terminal complexes (TCs) in algae are the structures that synthesize cellulose and secrete cellulose microfibrils. In the Zygnemataceae, each single rosette forms a 5-nm or 3-nm single “elementary” microfibril (primary wall), whereas rosettes arranged in rows of hexagonal arrays synthesize criss-crossed bands of parallel cellulose microfibrils (secondary wall). In Spirogyra, it is proposed that each of the six subunits of a rosette might synthesize six β-1,4-glucan chains that cocrystallize into a 36-glucan chain “elementary” microfibril, as is the case in higher plants. One typical feature of the linear terminal complexes in red algae is the periodic arrangement of the particle rows transverse to the longitudinal axis of the TCs. In bangiophyte red algae and in Vaucheria hamata, cellulose microfibrils are thin, ribbon-shaped structures, 1–1.5 nm thick and 5–70 nm wide; details of their synthesis are reviewed. Terminal complexes appear to be made in the endoplasmic reticulum and are transferred to Golgi cisternae, where the cellulose synthases are activated and may be transported to the plasma membrane. In algae with linear TCs, deposition follows a precise pattern directed by the movement and the orientation of the TCs (membrane flow). A principal underlying theme is that the architecture of cellulose microfibrils (size, shape, crystallinity, and intramicrofibrillar associations) is directly related to the geometry of TCs. The effects of inhibitors on the structure of cellulose-synthetizing complexes and the relationship between the deposition of the cellulose microfibrils with cortical microtubules and with the membrane-embedded TCs is reviewed In Porphyra yezoensis, the frequency and distribution of TCs reflect polar tip growth in the apical shoot cell.The evolution of TCs in algae is reviewed. The evidence gathered to date illustrates the utility of terminal complex organization in addressing plant phylogenetic relationships.     

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.     

Role of the putative membrane-bound endo-1,4-beta-glucanase KORRIGAN in cell elongation and cellulose synthesis in Arabidopsis thaliana

A temperature-sensitive, elongation-deficient mutant of Arabidopsis thaliana was isolated. At the non-permissive temperature of 31 degrees C, the mutation impaired tissue elongation; otherwise, tissue development was normal. Hypocotyl cells that had established cell walls at 21 degrees C under light-dark cycles ceased elongation and swelled when the mutant was shifted to 31 degrees C and darkness, indicating that the affected gene is essential for cell elongation. Analysis of the cell walls of mutant plants grown at 31 degrees C revealed that the cellulose content was reduced to 40% and the pectin content was increased to 162% of the corresponding values for the wild type grown at the same temperature. The increased amounts of pectin in the mutant were bound tightly to cellulose microfibrils. No change in the content of hemicellulose was apparent in the 31 degrees C-adapted mutant. Field emission-scanning electron microscopy suggested that the structure of cellulose bundles was affected by the mutation; X-ray diffraction, however, revealed no change in the crystallite size of cellulose microfibrils. The regeneration of cellulose microfibrils from naked mutant protoplasts was substantially delayed at 31 degrees C. The recessive mutation was mapped to chromosome V, and map-based cloning identified it as a single G-->A transition (resulting in a Gly(429)-->Arg substitution) in KORRIGAN, which encodes a putative membrane-bound endo-1,4-beta-glucanase. These results demonstrate that the product of this gene is required for cellulose synthesis.     

Cellulose synthesizing terminal complexes and morphogenesis in tip-growing cells of Syringoderma phinneyi (Phaeophyceae)

The intramembrane particles and cellulose synthesis of the brown alga Syringoderma phinneyi Henry et Müller were examined using replicas of freeze‐fractured apical cells. Like in other brown algae, linear terminal complexes (TCs) were found in the plasmatic fracture face (PF) of the plasmalemma, which are the putative cellulose synthases. Terminal complexes consist of a single row of particles, each particle composed of two sub‐units, and are found in close relationship with cellulose microfibril imprints. Examination of the distribution of TCs revealed a clear apico‐basal gradient, with a higher density of TCs in the apical part. This seems to reflect the tip growth of the apical cells. The rate of cellulose synthesis per TC subunit was calculated based on the dimensions of the TCs and cellulose microfibrils.     

Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.     

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.     

Cellulose microfibril assembly inErythrocladia subintegra Rosenv.: An ideal system for understanding the relationship between synthesizing complexes (TCs) and microfibril crystallization

Summary The marine red algaErythrocladia subintegra synthesizes cellulose microfibrils as determined by CBH I-gold labelling, X-ray and electron diffraction analyses. The cellulose microfibrils are quite thin, ribbon-like structures, 1–1.5 nm in thickness (constant), and 10–33 nm in width (variable). Several laterally associated minicrystal components contribute to the variation in microfibrillar width. Electron diffraction analysis suggested a uniplanar orientation of the microfibrils with their (101) lattice planes parallel to the plasma membrane surface of the cell. The linear particle arrays bound in the plasma membrane and associated with microfibril impressions recently demonstrated inErythrocladia have been shown in this study to be the cellulose-synthesizing terminal complexes (TCs). The TCs appear to be organized by a repetition of transverse rows consisting of four TC subunits, rather than by four rows of longitudinallyarranged TC subunits. The number of transverse rows varied between 8–26, corresponding with variation in the length of the TCs and the width of the microfibrils. The spacings between the neighboring transverse rows are almost constant being 10.5–11.5 nm. Based on the knowledge thatAcetobacter, Vaucheria, andErythrocladia synthesize similar thin, ribbon-like cellulose microfibrils, the structural characteristics common to the organization of distinctive TCs occurring in these three organisms has been discussed, so that the mode of cellulose microfibril assembly patterns may be deciphered.     

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).