Induction of multinucleation by β-glucosyl Yariv reagent in regenerated cells from <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> protoplasts and involvement of arabinogalactan proteins in cell plate formation

Arabinogalactan proteins (AGPs) are abundant plant cell surface proteoglycans widely distributed in plant species. Since high concentrations of β-glucosyl Yariv reagent (βglcY), which binds selectively to AGPs, inhibited cell division of protoplast-regenerated cells of the liverwort Marchantia polymorpha L. (Shibaya and Sugawara in Physiol Plant 130:271–279, 2007), we investigated the mechanism underlying the inability of the cells to divide normally by staining nuclei, cell walls and β-1,3-glucan. Microscopic observation showed that the diameter of regenerated cells cultured with βglcY was about 2.8-fold larger than that of cells cultured without βglcY. The cells cultured with βglcY were remarkably multinucleated. These results indicated that βglcY did not inhibit mitosis but induced multinucleation. In the regenerated cells cultured with low concentrations of βglcY (5 and 1 μg ml⁻¹), the cell plate was stained strongly by βglcY, suggesting abundant AGPs in the forming cell plate. In these cell plates, β-1,3-glucan was barely detectable or not detected. In multinucleated cells, cell plate-like fragments, which could not reach the cell wall, were frequently observed and they were also stained strongly by βglcY. Our results indicated that AGPs might have an important role in cell plate formation, and perturbation of AGPs with βglcY might result in remarkable multinucleation in protoplast-regenerated cells of M. polymorpha. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A role for arabinogalactan-proteins in root epidermal cell expansion

Arabinogalactan-proteins (AGPs) are abundant plant proteoglycans that react with (β-d-Glc)₃ but not (β-d-Man)₃ Yariv reagent. We report here that treatment with (β-d-Glc)₃ Yariv reagent caused inhibition of root growth of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, the treated roots exhibited numerous bulging epidermal cells. Treatment with (β-d-Man)₃ Yariv reagent did not have any such effects. These results indicate a role for AGPs in root growth and control of epidermal cell expansion. Because treatment with (β-d-Glc)₃ Yariv reagent phenocopies the reb1 (root epidermal cell bulging) mutant of Arabidopsis, AGPs were extracted from the reb1-1 mutant and compared with those of the wild type. The reb1-1 roots contained an approximately 30% lower level of AGPs than the wild type. More importantly, while the profile of AGPs from wild-type roots showed two major peaks upon crossed electrophoresis, the profile of AGPs from reb1-1 roots exhibited only one of the major peaks. Therefore, the reb1 phenotype appears to be a result of defective or missing root AGPs. Taken together, this pharmacological and genetic evidence strongly indicates a function of AGPs in the control of root epidermal cell expansion. Received: 13 February 1997 / Accepted: 1 April 1997     

Arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of Euphorbia pulcherrima

The arabinogalactan protein (AGP) fractions of embryogenic and non-embryogenic callus lines of Euphorbia pulcherrima Willd. ex. Klotzsch were analysed over a cultivation period of 9 weeks using the β -glucosyl Yariv reagent and an anti-AGP antibody (LM2). The amount of AGPs detected with the Yariv reagent increased in embryogenic cultures during the development of somatic embryos. The embryogenic and non-embryogenic callus contained different sets of AGPs characterized with the Yariv reagent and the LM2 monoclonal antibody. AGPs recognized by LM2 are localized primarily in the protodermal cells of globular somatic embryos. The development of somatic embryos of E. pulcherrima appears to be associated with the presence of particular AGPs.     

Purification and characterization of wall-localized cellulase from maize coleoptiles

Wall-localized cellulase was partially purified from freeze-dried maize coleoptiles by a combination of DEAE-Sepharose, Superdex-200 gel filtration and Hydroxyapatite column chromatography. Activity was measured by both reducing sugar assay and dot assay on agarose gel containing carboxymethylcellulose(CMC). In situ activity staining on a nondenaturing gel overlaid on agarose gel containing CMC turned out to be a quite reliable method to detect cellulase activity. The molecular mass of partially-purified cellulase was determined to be about 53 kD based on SDS-PAGE, and the N-terminal amino acid sequence of this cellulase was NH₂-AGAKGANXLGGLXRA. The enzyme hydrolyzed CMC with an optimal pH of 4.5 and optimal temperature of 40°C. It also catalyzed carboxymethylcellulose with aK _m of 2.02 mg/mL and aV _max of 160 ng/h/mL The β-1,4-glucosyl linkages of CMC, fibrous cellulose and lichenan were cleaved specifically by this enzyme. Reducing reagents such as cysteine-HCI, dithiothreitol and glutathione strongly enhanced the activity, suggesting that SH-groups of the enzyme were protected from oxidation. N-ethylmaleimide which is a sulfhydryl-reacting reagent did not seem to inhibit the activity, indicating that cysteine residues were not located near the active site of the enzyme. These results will be valuable in understanding the structure of wall-localized cellulase in maize coleoptiles and in predicting its possible function in the cell wall.     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

Stage-specific responses of embryogenic carrot cell suspension cultures to arabinogalactan protein-binding β-glucosyl Yariv reagent

The arabinogalactan protein-binding β-d-glucosyl Yariv reagent (βGlcY) was applied to the various developmental stages of embryogenic carrot (Daucus carota L. cv. Early Nantes) cell-suspension cultures. Roots without shoot structures were produced in cultures grown under embryo-inducing conditions in medium containing βGlcY. Only low concentrations of βGlcY permitted the subsequent production of embryos in these cultures. When early stage embryos were transferred to medium containing βGlcY, the roots elongated greatly while the shoot apices expanded radially. These embryos did not progress to the next developmental stage. Torpedo embryos and plantlets, however, showed an overall inhibition of growth in the presence of βGlcY. Developmental stage therefore appears to determine how cultures and embryos respond to βGlcY, root growth being promoted in the early stages, and overall growth reduced in the late stages. Received: 1 July 1997 / Accepted: 31 July 1997     

Localization of arabinogalactan-proteins in different stages of embryos and their role in cotyledon formation of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Arabinogalactan proteins (AGPs) have been implicated in plant development including sexual plant reproduction. In this paper, the expression of AGPs and the effects of β-glucosyl Yariv reagent (βGlcY, which binds arabinogalactan proteins) in embryo development and cotyledon formation were investigated. Immunofluorescence assay displayed that the expression of AGPs labeled with antibody JIM13 was developmentally regulated. In early stages, AGPs were evenly distributed in the whole embryo, except for a short polar expression in the basal suspensor cell. In the globular stage of embryo, AGPs were condensed in the embryo proper (EP), apex of the EP, and at the juncture of the EP and suspensor. In heart-shaped embryo, APGs were only present at the juncture of the EP and suspensor. Immunogold labeling assay showed that the strong expression of AGPs at the juncture of the EP and suspensor was localized in the cell wall. Provision of βGlcY to the in vitro ovule culture medium caused delayed growth of embryos, cotyledon defect and abnormal venation pattern. Consequently, βGlcY induced the death of defective seedlings with the characteristics of deformed or irregular single cotyledon. Our results suggested that AGPs play functional roles in embryo development, cotyledon formation and seedling morphology establishment in Nicotiana tabacum L.     

Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and development. (β-d-glucosyl)₃ Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50 μM β-GlcY. However, the growth was not inhibited by (α-d-galactosyl)₃ Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis.     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (βglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml⁻¹ inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of β-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.     

An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine

Arabinogalactan proteins (AGPs) are abundant plant proteoglycans implicated in plant growth and development. Here, we report the genetic characterization, partial purification and immunolocalization of a classical AGP (PtaAGP6, accession number AF101785) in loblolly pine (Pinus taeda L.). A PtaAGP6 full-length cDNA clone was expressed in bacteria. PtaAGP6 resembles tomato LeAGP-1 and Arabidopsis AtAGP17-19 in that they all possess a subdomain composed of basic amino acids. The accessibility of this domain in the glycoprotein makes it possible to label the PtaAGP6 epitopes on the cell surface or in the cell wall with polyclonal antibodies raised against this subdomain. The antibodies recognize the peptide of the basic subdomain and bind to the intact protein molecule. A soluble protein-containing fraction was purified from the differentiating xylem of pine trees by using -glucosyl Yariv reagent (-glcY) and was recognized by antibodies against the basic subdomain. Immunolocalization studies showed that the PtaAGP6 epitopes are restricted to a file of cells that just precede secondary cell wall thickening, suggesting roles in xylem differentiation and wood formation. The location of apparent labeling of the PtaAGP6 epitopes is separated from the location of lignin deposition. Multiple single nucleotide polymorphisms (SNPs) were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that PtaAGP6 is encoded by a single gene. Mobility variation in denaturing gel electrophoresis was used to map PtaAGP6 SNPs to a site on linkage group 5.     

Disruption of arabinogalactan proteins disorganizes cortical microtubules in the root of Arabidopsis thaliana

The cortical array of microtubules inside the cell and arabinogalactan proteins on the external surface of the cell are each implicated in plant morphogenesis. To determine whether the cortical array is influenced by arabinogalactan proteins, we first treated Arabidopsis roots with a Yariv reagent that binds arabinogalactan proteins. Cortical microtubules were markedly disorganized by 1 microM beta-D-glucosyl (active) Yariv but not by up to 10 microM beta-D-mannosyl (inactive) Yariv. This was observed for 24-h treatments in wild-type roots, fixed and stained with anti-tubulin antibodies, as well as in living roots expressing a green fluorescent protein (GFP) reporter for microtubules. Using the reporter line, microtubule disorganization was evident within 10 min of treatment with 5 microM active Yariv and extensive by 30 min. Active Yariv (5 microM) disorganized cortical microtubules after gadolinium pre-treatment, suggesting that this effect is independent of calcium influx across the plasma membrane. Similar effects on cortical microtubules, over a similar time scale, were induced by two anti-arabinogalactan-protein antibodies (JIM13 and JIM14) but not by antibodies recognizing pectin or xyloglucan epitopes. Active Yariv, JIM13, and JIM14 caused arabinogalactan proteins to aggregate rapidly, as assessed either in fixed wild-type roots or in the living cells of a line expressing a plasma membrane-anchored arabinogalactan protein from tomato fused to GFP. Finally, electron microscopy of roots prepared by high-pressure freezing showed that treatment with 5 microM active Yariv for 2 h significantly increased the distance between cortical microtubules and the plasma membrane. These findings demonstrate that cell surface arabinogalactan proteins influence the organization of cortical microtubules.     

Histochemistry and composition of the cell walls of styles of Nicotiana alata Link et Otto

The cell walls of styles of Nicotiana alata Link et Otto (ornamental tobacco; Solanaceae) were analysed chemically and examined histochemically. Cell-wall preparations were obtained from whole styles and from isolated transmitting-tissue cells. The style epidermal cells were shown histochemically to have thick, lignified secondary walls. These walls probably constituted a large proportion of the cell-wall preparation from whole styles as analysis of whole-style walls indicated that the major polysaccharides were xylans and cellulose, which are typical of lignified secondary walls of Magnoliopsida (dicotyledons). Lignification of the style epidermal walls was also demonstrated histochemically in 10 other species (5 genera including Nicotiana) of the sub-family Cestroideae of the Solanaceae, but not in 15 species (9 genera) of the sub-family Solanoideae of the Solanaceae, nor in 3 other species of dicotyledons and 2 species of Liliopsida (monocotyledons). Analysis of the cell-wall preparation from isolated transmitting-tissue cells of N. alata indicated that these contained cellulose, xyloglucans, and pectic polysaccharides, which is typical of primary cell walls of dicotyledons. However, the analysis indicated that the walls also contained an unusually high proportion of Type II arabinogalactans. Staining of the transmitting-tissue cell-wall preparation with β-glucosyl Yariv reagent, a histochemical reagent specific for arabinogalactan proteins, confirmed their presence, which may be related to the role of these cells in secreting the stylar extracellular matrix.     

Immunochemical comparison of membrane-associated and secreted arabinogalactan-proteins in rice and carrot

Arabinogalactan-proteins (AGPs) occurring in suspension-cultured rice (Oryza saliva L.) cells, their conditioned medium and at the rice root apex were investigated using monoclonal antibodies and the AGP-binding -glucosyl Yariv reagent ( GlcY). A monoclonal antibody, LM2, was generated that recognized an acidic carbohydrate epitope common to two soluble AGPs occurring in the conditioned medium of proliferating rice cells, membrane-associated AGPs (rmAGP) in the cultured cells and two AGPs at the rice root apex. In addition, LM2 recognized AGPs secreted by suspensioncultured carrot (Daucus carota L.) cells. The two AGPs of the rice culture medium, srAGP1 and srAGP2, were discriminated by their mobilities during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reaction with GlcY, the presence of arabinogalactan epitopes and anion-exchange chromatography. The association of rmAGP with the plasma membrane was investigated by Triton-X-114/aqueous partitioning of both microsomal and plasma-membrane preparations and rmAGP was found to partition into the detergent phase, indicating that AGPs are hydrophobic plasma-membrane proteins in rice. This was in contrast to plasma-membrane AGPs of suspension-cultured carrot cells that partitioned into the aqueous phase. At the rice root apex most of the AGP was associated with the microsomal fraction and also partitioned into the detergent phase, although a distinct highmolecular-mass AGP entered the aqueous phase.Abbreviations AGP arabinogalactan-protein - GlcY -glucosyl Yariv reagent - ELISA enzyme-linked immunosorbent assay We gratefully acknowledge support from the Leverhulme Trust, the UK Biotechnology and Biological Sciences Research Council and the Royal Society.     

Protein expression during seed development in Araucaria angustifolia: transient accumulation of class IV chitinases and arabinogalactan proteins

Protein accumulation and patterns during embryogenesis in the recalcitrant seeds of the gymnosperm species Araucaria angustifolia (Bert.) O. Kuntze were studied. Soluble seed proteins, chitinases, and arabinogalactan proteins (AGPs) were analyzed by means of 2-D gel electrophoresis, mass spectrometry, isoelectric focusing, Western blot, precipitation and staining with β-glucosyl Yariv reagent (β-Glc)₃Y, and gas liquid chromatography. Despite the recalcitrant nature of the seeds, the electrophoretic patterns of A . angustifolia seed proteins showed similarities with orthodox seed types. Proteins showing chitinolytic activity were observed in all seed stages analyzed, but the expression of class IV chitinases was restricted to late stages of seed development. AGPs were prominent during stages of seed development characterized by intensive cell division and differentiation, and their decrease during seed maturation might be related to cell wall modifications during the deposition of storage compounds. Gas liquid chromatographic analyzes of AGPs did not show quantitative changes in their carbohydrate moieties during seed development. A low molecular weight protein specifically expressed in mature seeds was precipitated using (β-Glc)₃Y. Amino acid sequences obtained from MS/MS analysis revealed peptides rich in valine and acidic amino acids, but devoid in amino acids normally found in AGPs polypeptides, suggesting that these peptides are not related to classical or non-classical AGPs. Possible implications of chitinases and AGPs during seed development in A . angustifolia are discussed.     

Production of cellulase by a wild strain of Chaetomium globosumusing delignified oil palm empty-fruit-bunch fibre as substrate

Studies on the feasibility of using delignified oil palm empty-fruit-bunch (OPEFB) fibres as a substrate for cellulase production by Chaetomium globosum strain 414 were carried out in shake-flask cultures containing different types and concentrations of nitrogen source. Peptone, as nitrogen source, gave maximum production of all the three main components of the cellulase complex (endoglucanase or carboxymethylcellulase, cellobiohydrolase or filter-paper-hydrolysing enzyme and β-glucosidase), followed by yeast extract, urea, KNO₃ and (NH₄)₂SO₄. The maximum specific growth rate (μ_max) of C. globosum strain 414 grown in medium containing OPEFB and peptone was 0.038 h⁻¹. In all the fermentations, the fungus was able to produce all the three cellulases with significant amounts of β-glucosidase, except when using (NH₄)₂SO₄ as nitrogen source, where β-glucosidase was not produced. With 6 g/l peptone and 10 g/l delignified OPEFB fibres, the fungus produced maximum concentrations of FPase, carboxymethylcellulase and β-glucosidase: 1.4, 30.8 and 9.8 U/ml, giving productivities of 10, 214 and 24 U l⁻¹h⁻¹, respectively. The cellulase mixture, partially purified by ammonium sulphate precipitation, was able to hydrolyse delignified OPEFB fibres, converting about 68 % of the cellulosics to reducing sugars after 5 days. Received: 17 June 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of β-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana

Seedlings of Arabidopsis thaliana were germinated and grown in medium containing β-glucosyl Yariv reagent (βGlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of βGlcY on the seedlings was to reduce the overall growth of both the root and the shoot. βGlcY only accumulated in the root tissues and the reduced growth of the shoot appeared to be an indirect effect of impaired root growth. Reduced root growth was a consequence of a reduction in cell elongation during the postproliferation phase of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. βGlcY penetrated roots as far as the endodermis and it is suggested that the interaction of βGlcY with AGPs in the load-bearing cell layers inhibited root elongation. When βGlcY was added to carrot suspension-cultured cells that had been induced to elongate rather than proliferate, cell elongation was inhibited. The AGP-unreactive α-galactosyl Yariv reagent (αGalY) had no biological activity in either of these systems.     

Secondary cell-wall assembly in flax phloem fibres: role of galactans

Non-lignified fibre cells (named gelatinous fibres) are present in tension wood and the stems of fibre crops (such as flax and hemp). These cells develop a very thick S2 layer within the secondary cell wall, which is characterised by (1) cellulose microfibrils largely parallel to the longitudinal axis of the cell, and (2) a high proportion of galactose-containing polymers among the non-cellulosic polysaccharides. In this review, we focus on the role of these polymers in the assembly of gelatinous fibres of flax. At the different stages of fibre development, we analyse in detail data based on sugar composition, linkages of pectic polymers, and immunolocalisation of the β-(1→4)-galactans. These data indicate that high molecular-mass gelatinous galactans accumulate in specialised Golgi-derived vesicles during fibre cell-wall thickening. They consist of RG-I-like polymers with side chains of β-(1→4)-linked galactose. Most of them are short, but there are also long chains containing up to 28 galactosyl residues. At fibre maturity, two types of cross-linked galactans are identified, a C–L structure that resembles the part of soluble galactan with long side chains and a C–S structure with short chains. Different possibilities for soluble galactan to give rise to C–L and C–S are analysed. In addition, we discuss the prospect for the soluble galactan in preventing the newly formed cellulose chains from completing immediate crystallisation. This leads to a hypothesis that firstly the secretion of soluble galactans plays a role in the axial orientation of cellulose microfibrils, and secondly the remodelling and cross-linking of pectic galactans are linked to the dehydration and the assembly of S2 layer.     

Protein changes during programmed cell death in tobacco

Programmed cell death (PCD) was induced by the Yariv reagent in Nicotiana tabacum cv. Bright Yellow-2 cell suspension. The analyses of proteins extracts by 2-D electrophoresis clearly show massive protein degradation which was mainly due to cysteine protease activity. In contrast, some proteins remained unchanged up to 72 h after PCD induction. Peptide mass fingerprints of these proteins, obtained by MALDI-TOF, identified calreticulin, heat shock protein (HSP) 60, HSP70, malate dehydrogenase and mitochondrial ATP synthase β-subunit.