3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.     

Sequential deposition of plant glycoproteins and polysaccharides at the host-parasite interface ofUromyces vignae andVigna sinensis

Summary Monokaryotic haustoria (M-haustoria) ofUromyces vignae inVigna sinensis cells are surrounded by an extrahaustorial matrix (ema) and the invaginated host plasmalemma, the extrahaustorial membrane (ehrn). The ema was characterized with antibodies against components of the plant cell wall; the ema contained hydroxyproline-rich glycoproteins and arabinogalactans/arabinogalactan proteins, both at a higher concentration close to the ehm. Haustoria with large vacuoles had the ema encased by additional layers. An electron-translucent inner layer deposited on top of the ema contained arabinogalactans/arabinogalactan proteins, hydroxyproline-rich glycoproteins, and callose. The inner layer was surrounded by an electron-translucent middle layer with numerous dark inclusions, rich in pectin and fucose bound to xyloglucans. Finally, a more electron-dense outer layer containing arabinogalactans/arabinogalactan proteins and hydroxyproline-rich glycoproteins encased the whole structure. These polysaccharides, with the exception of callose and un-esterified pectin, were also found in the plant Golgi apparatus. The polysaccharides were synthesized in the trans Golgi cisternae and secreted into the host-parasite interface. The secretory events seem to be coupled to endocytosis since numerous coated pits were found on the ehm too. The pits were elongated, sometimes formed tubules and the coat reacted with an antibody against plant clathrin. Our results suggest intensive membrane recycling around haustoria, together with the secretion of cell wall material, which in the case of more or less vacuolated haustoria seems to be responsible for encasementAbbreviations AG/AGP arabinogalactans and arabinogalactan proteins - BSA bovine serum albumin - ehm extrahaustorial membrane - ema extrahaustorial matrix - HRGP_2b hydroxyproline rich glycoproteins - M-haustorium monokaryotic haustorium - TBS tris buffered saline     

THE ULTRASTRUCTURE OF THE PELLICLE COMPLEX OF EUGLENA GRACILIS

The pellicle complex of E. gracilis is composed of the cell membrane, the ridge and groove with the notch, four fibrils, and the subpellicular ER. The cell membrane is of unit membrane configuration and covers the outside of the cell, the cytostome, the gullet, and the reservoir. The notch of the pellicle complex has always a close topographic relationship to two particular fibrils, as well as the subpellicular ER. The gullet is that region between the reservoir and the cytostome which, in addition to longitudinal fibrils, is surrounded by a single row of circular fibrils. The circumference of the cytostome has twenty large pellicular ridges alternating with small pellicular ridges. Alternating tall and small pellicular ridges cover the entire cell during division.     

植物伸展蛋白的研究进展

伸展蛋白是高等植物细胞壁中一族富含羟脯氨酸的糖蛋白,在植物细胞壁中发挥着重要的生理功能。综述了近几十年对伸展蛋白结构、功能、基因家族以及生物合成与基因表达调节的研究进展。     

植物细胞壁中的伸展蛋白

随着实验技术的发展尤其是分子生物学技术的应用 ,植物细胞壁的研究已取得丰硕的成果。植物细胞壁中最重要的结构蛋白———伸展蛋白 ,是高等植物细胞壁中一族富含羟脯氨酸的糖蛋白 ,起强固细胞壁的作用。本文综述了近几十年对伸展蛋白的分离纯化、结构、生物合成、功能作用及其基因和表达的控制方面的研究     

Plant Cell Wall Remodelling in the Rhizobium–Legume Symbiosis

Colonization of host cells by rhizobium bacteria involves the progressive remodelling of the plant–microbial interface. Following induction of nodulation genes by legume-derived flavonoid signals, rhizobium secretes Nod-factors (lipochitin oligosaccharides) that cause root hair deformations by perturbing the growth of the plant cell wall. The infection thread arises as a tubular ingrowth bounded by plant cell wall. This serves as a conduit for colonizing bacterial cells that grow and divide in its lumen. The transcellular orientation of thread growth is controlled by the cytoskeleton and is coupled to cell cycle reactivation and cell division processes. In response to rhizobium infection, host cells synthesize several new components (early nodulins) that modify the properties of the cell wall and extracellular matrix. Root nodule extensins are a legume-specific family of hydroxyproline-rich glycoproteins targeted into the lumen of the infection thread. They have alternating extensin and arabinogalactan (AGP) glycosylation motifs. The structural characteristics of these glycoproteins suggest that they may serve to regulate fluid-to-solid transitions in the extracellular matrix. Extensibility of the infection thread is apparently controlled by peroxide-driven protein cross-linking and perhaps also by modification of the pectic matrix. Endocytosis of rhizobia from unwalled infection droplets into the host cell cytoplasm depends on physical contact between glycocalyx components of the plant and bacterial membrane surfaces. As endosymbionts, bacteroids remain enclosed within a plant-derived membrane that is topologically equivalent to the plasma membrane. This membrane acquires specialist functions that regulate metabolite exchanges between bacterial cells and the host cytoplasm. Ultimately, however, the fate of the symbiosome is to become a lysosome, causing the eventual senescence of the symbiotic interaction.     

Isodityrosine cross-linking mediates insolubilization of cell walls in Chlamydomonas.

Enzymatic removal of the cell wall induces vegetative Chlamydomonas reinhardtii cells to transcribe wall genes and synthesize new hydroxyproline-rich glycoproteins (HRGPs) related to the extensins found in higher plant cell walls. A cDNA expression library made from such induced cells was screened with antibodies to an oligopeptide containing the (SP)x repetitive domains found in Chlamydomonas wall proteins. One of the selected cDNAs encodes an (SP)x-rich polypeptide that also displays a repeated YGG motif. Ascorbate, a peroxidase inhibitor, and tyrosine derivatives were shown to inhibit insolubilization of both the vegetative and zygotic cell walls of Chlamydomonas, suggesting that oxidative cross-linking of tyrosines is occurring. Moreover, insolubilization of both walls was concomitant with a burst in H2O2 production and in extracellular peroxidase activity. Finally, both isodityrosine and dityrosine were found in hydrolysates of the insolubilized vegetative wall layer. We propose that the formation of tyrosine cross-links is essential to Chlamydomonas HRGP insolubilization.     

STUDIES ON THE BIOSYNTHESIS OF COLLAGEN : I. THE GROWTH OF FOWL OSTEOBLASTS AND THE FORMATION OF COLLAGEN IN TISSUE CULTURE

1. A tissue culture method was devised in which suspensions of osteoblasts, obtained directly from frontal bones of fowl embryos, were grown in a fluid, fibrin-free medium. 2. Maximum growth of the tissue, as measured by dry weight, with the formation of collagen protein, based on the estimation of hydroxyproline, was obtained in periods of up to 6 days. 3. Appreciable amounts of protein-bound hydroxyproline were formed during the first 24 hour growth period, but electron microscopy of portions of the same cultures failed to demonstrate the presence of any typical collagen fibrils. 4. The subsequent formation of many characteristic collagen fibrils was not associated with a significant rise in the mean hydroxyproline content of the tissue. 5. The cytoplasmic granules of the osteoblasts stained intensely with the P.A.S. technique when the collagen fibrils were being formed. 6. It is suggested that collagen-forming cells synthesise and secrete a hydroxyproline-rich precursor of protein or large peptide nature, which subsequently becomes directly transformed into typical collagen fibrils.     

THE PRESENCE OF CALLOSE IN THE PRIMARY ZYGOTE WALL OF CHLAMYDOMONAS MONOICA AND THE EFFECTS OF ITS DEGRADATION ON ZYGOTE DEVELOPMENT

Chlamydomonas monoica constructs a temporary primary wall around its developing zygotes. This study aimed to confirm callose as a component of the primary wall, as well as to note the effects of primary wall degradation on zygote development. Glucanase, specific for the β-1,3 glycosidic bonds comprising callose, was added to mating media at concentrations ranging from 5 to 1 mg ml⁻¹ and light microscope observations were made as the zygotes developed. The overall health of the zygotes was assessed by comparing their ability to germinate after exposure to chloroform vapors. The bright staining of the primary wall with aniline blue, specific for β-1,3 polysaccharides, suggested the presence of callose. This was further supported by the adverse effects of glucanase on zygote development. After mating, declining levels of intact zygotes were found as their maturation continued, and dead immature zygotes accumulated in the treated cultures. Twelve days after mating, when the zygotes were plated for germination, fully mature zygotes were identified in only the lowest of the six enzyme concentrations. In addition, germinating zygotes from the treated cultures showed increased sensitivity to killing by chloroform vapors relative to untreated zygotes. These results suggest that callose is a key component in the primary zygote wall, and that its degradation negatively affects zygote maturation. Electron microscopy will be used to help determine whether structural defects in the primary wall occur as a result of glucanase treatment, and whether such defects affect secondary zygospore wall assembly.     

Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp₄ glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.     

ELECTRON-OPAQUE FIBRILS AND GRANULES IN AND BETWEEN THE CELL WALLS OF HIGHER PLANTS

The components of higher-plant cell walls which become electron-opaque after staining with ruthenium-osmium were studied by electron microscopy. A fibrillar material which absorbs this stain is a major wall constituent in the root epidermal cells of carrot and morning glory. In both form and size, these fibrils resemble those found on the surface of suspension-cultured cells of the same species Some cells of woody species show an irregular distribution of electron-opaque material in the cell wall matrix and middle lamella. This material, which has an amorphous appearance with many electron stains, is shown by ruthenium-osmium staining to be an aggregate of discrete granules, 150–220 A in diameter. These observations are not consistent with the concept of the cell wall matrix and middle lamella as an amorphous, uniform gel     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

Molecular characterization of a zygote wall protein: an extensin-like molecule in Chlamydomonas reinhardtii.

The green alga Chlamydomonas reinhardtii elaborates two biochemically and morphologically distinct cell walls during its life cycle: one surrounds the vegetative and gametic cell and the other encompasses the zygote. Hydroxyproline-rich glycoproteins (HRGPs) constitute a major component of both walls. We describe the isolation and characterization of a zygote-specific gene encoding a wall HRGP. The derived amino acid sequence of this algal HRGP is similar to those of higher plant extensins, rich in proline and serine residues and possessing repeating amino acid motifs, notably X(Pro)3 and (Ser-Pro)n. Antiserum against this zygote wall protein detected common epitopes in several other zygote polypeptides, at least one of which is also encoded by a zygote-specific gene. We conclude that there is one set of HRGP wall genes expressed only in zygotes and another set that is specific to vegetative and gametic cells.     

Proteomic analysis of high-CO(2)-inducible extracellular proteins in the unicellular green alga, Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a wide range of CO(2) concentrations through the regulation of a CO(2)-concentrating mechanism (CCM). By proteomic analysis, here we identified the proteins which were specifically accumulated under high-CO(2) conditions in a cell wall-less strain of C. reinhardtii which release their extracellular matrix into the medium. When the CO(2) concentration was elevated from the ambient air level to 3% during culture, the algal growth rate increased 1.5-fold and the composition of extracellular proteins, but not intracellular soluble and insoluble proteins, clearly changed. Proteomic analysis data showed that the levels of 22 of 129 extracellular proteins increased for 1 and 3 d and such multiple high-CO(2)-inducible proteins include gametogenesis-related proteins and hydroxyproline-rich glycoproteins. However, we could not prove the induction of gametogenesis under high-CO(2) conditions, suggesting that the inductive signal might be incomplete, not strong enough or that only high-CO(2) conditions might be not sufficient for the cell stage to proceed to the formation of sexually active gametes. However, these gametogenesis-related proteins and/or hydroxyproline-rich glycoproteins may have novel roles outside the cell under high-CO(2) conditions.     

Extensin—a major cell wall glycoprotein

Abstract The structure of extensin is described in detail. It has a hydroxyproline-rich backbone, which contains repeating peptides glycosylated by short side chains and it adopts a polyproline II helical conformation. The glycoprotein is synthesized intracellularly and soluble precursors are secreted to the wall, where they are bound, perhaps, by the formation of isodityrosine cross-links. The various hypotheses, including the most recent ‘warp and weft’ model, which have been suggested to explain the attachment of extensin to the other wall polymers are discussed. The possible functions of extensin in defence and in the control of extension growth are described in addition to its probable structural role. Other glycoproteins which resemble extensin are also mentioned.     

THE OCCURRENCE AND PHYLOGENETIC SIGNIFICANCE OF PUTATIVE PLACENTAL TRANSFER CELLS IN THE GREEN ALGA COLEOCHAETE

Following fertilization, zygotes of the green alga Coleochaete orbicularis, which are retained on the haploid thallus, first enlarge, then become covered with a layer of vegetative cells. Light microscopy and high-voltage electron microscopy revealed the presence of localized wall ingrowths in vegetative cells adjacent to zygotes. These covering cells resemble the gametophytic placental transfer cells of embryophytes in their morphology, location, and time of development. If Coleochaete cells with wall protuberances function as do placental transfer cells of embryophytes, their presence is evidence that photosynthates may be transported between haploid thallus cells and zygotes. Thus, a nutritional relationship between different phases of the life cycle, similar to that which occurs in embryophytes, may also have evolved in green algae. This first report of putative placental transfer cells in a green alga supports Bower's (1908) ideas concerning the origin of land plant sporophytes and alternation of generations. The presence or absence of cells with wall ingrowths in several species of Coleochaete was correlated with estimates of zygote-plant area ratios.     

FEM1, a Fusarium oxysporum glycoprotein that is covalently linked to the cell wall matrix and is conserved in filamentous fungi

As part of an investigation of the cell wall structure of plant pathogenic, filamentous fungi, we set out to characterize covalently bound cell wall glycoproteins (CWPs) of the tomato pathogen Fusarium oxysporum. N-terminal sequencing of an abundant 60-kDa CWP led to the cloning of the corresponding gene, which we have designated FEM1 (Fusarium extracellular matrix protein). The gene contains an ORF encoding a primary translation product of 212 amino acids, including an N-terminal 17-amino acid secretion signal sequence. Furthermore, FEM1p contains two potential N-glycosylation sites, and is rich in serine and threonine residues (29%) that could serve as O-glycosyl addition sites. At its C-terminus the protein contains a 22-amino acid sequence with the characteristics of a glycosyl-phosphatidylinositol (GPI) anchor addition signal. A mutant FEM1 protein lacking this GPI anchor addition signal is not retained in the fungal cell wall but released into the culture medium, indicating that in the wild-type protein this sequence functions to anchor the protein to the extracellular matrix. Southern analysis shows that FEM1 is present as a single-copy gene in all formae speciales of F. oxysporum tested and in F. solani. Database searches show that FEM1p homologous sequences are present in other filamentous fungi as well.     

阔口尖毛虫形成包囊期间细胞超微结构的观察

阔口尖毛虫形成囊期间,细胞质内出现条带状或管产产的内质网和由不同大小的囊泡组成的包囊壁前体。并且,前体的产生与内质网有关;细胞质内发生自噬泡消化现象,这是细胞将原有结构和能量进行贮存,利用的一种重要形式;大核向细胞质突出形成阿米巴形结构,这与大核向细胞质排出部分核物质有关。     

THE FINE STRUCTURE OF CORTICAL COMPONENTS OF PARAMECIUM MULTIMICRONUCLEATUM

The electron microscope was used to study the structure and three dimensional relationships of the components of the body cortex in thin sections of Paramecium multimicronucleatum. Micrographs of sections show that the cortex is covered externally by two closely apposed membranes (together ~250 A thick) constituting the pellicle. Beneath the pellicle the surface of the animal is molded into ridges that form a polygonal ridgework with depressed centers. It is these ridges that give the surface of the organism its characteristic configuration and correspond to the outer fibrillar system of the light microscope image. The outer ends of the trichocysts with their hood-shaped caps are located in the centers of the anterior and posterior ridges of each polygon. The cilia extend singly from the depressed centers of the surface polygons. Each cilium shows two axial filaments with 9 peripheral and parallel filaments embedded in a matrix and the whole surrouned by a thin ciliary membrane. The 9 peripheral filaments are double and these are evenly spaced in a circle around the central pair. The ciliary membrane is continuous with the outer member of the pellicular membrane, whereas the plasma membrane is continuous with the inner member of the pellicular membrane. At the level of the plasma membrane the proximal end of the cilium is continuous with its tube-shaped basal body or kinetosome. The peripheral filaments of the cilium, together with the material of cortical matrix which tends to condense around them, form the sheath of the basal body. The kinetodesma connecting the ciliary kinetosomes (inner fibrillar system of the light microscopist) is composed of a number of discrete fibrils which overlap in a shingle-like fashion. Each striated kinetosomal fibril originates from a ciliary kinetosome and runs parallel to other kinetosomal fibrils arising from posterior kinetosomes of a particular meridional array. Sections at the level of the ciliary kinetosomes reveal an additional fiber system, the infraciliary lattice system, which is separate and distinct from the kinetodesmal system. This system consists of a fibrous network of irregular polygons and runs roughly parallel to the surface of the animal. Mitochondria have a fine structure similar in general features to that described for a number of mammalian cell types, but different in certain details. The structures corresponding to cristae mitochondriales appear as finger-like projections or microvilli extending into the matrix of the organelle from the inner membrane of the paired mitochondrial membrane. The cortical cytoplasm contains also a particulate component and a system of vesicles respectively comparable to the nucleoprotein particles and to the endoplasmic reticulum described in various metazoan cell types. An accessory kinetosome has been observed in oblique sections of a number of non-dividing specimens slightly removed from the ciliary kinetosome and on the same meridional line as the cilia and trichocysts. Its position corresponds to the location of the kinetosome of the newly formed cilium in animals selected as being in the approaching fission stage of the life cycle.