Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans

 Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na₂CO₃, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides. Received: 29 May 1999 / Accepted: 19 October 1999     

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells

 A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [¹⁴C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent K _m of 35 μM and an apparent V _max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass >500 kDa. Received: 3 July 1999; Accepted: 27 September 1999     

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtiiy1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins. Received: 29 September 1999 / Accepted: 20 November 1999     

Studies of aminochrome toxicity in a mouse derived neuronal cell line: is this toxicity mediated via glutamate transmission?

Summary. Aminochrome was found to be toxic in a mouse-derived neuronal cell line (CNh). The effect was concentration dependent (10–150 μM). The issue whether aminochrome toxicity involves glutamate transmission was studied with several glutamate receptors antagonists. Incubation of the cells with aminochrome (150 μM) in the presence of 100 μM of the AMPA an-tagonist, NBQX resulted in an increase of cell survival, from 52 to 73%. However, this protective effect did not seem to be related to activation of ionotropic glutamate receptors since incubation of CNh cells with 200 μM of glutamate resulted in only 10% decrease of cell survival. However, NBQX was found to inhibit in vitro the autoxidation process. One hundred μM AP-5 did not have any effect on aminochrome toxicity. The toxic effect of aminochrome on CNh cells seems to be dependent of extracellular activation since addition of dicoumarol, a specific inhibitor of DT-diaphorase, did not affect that toxicity, which can be explained perhaps by a lack of a transport system for aminochrome into the CNh cells. Received July 28, 1999, Accepted December 6, 1999     

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives

 The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. × P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours. Received: 12 June 1999 / Accepted: 20 October 1999     

Immunolocalization of LeAGP-1, a modular arabinogalactan-protein, reveals its developmentally regulated expression in tomato

 Arabinogalactan-proteins (AGPs) are highly glycosylated cell surface proteins that are thought to function in plant growth and development. The developmentally regulated expression of LeAGP-1, a novel and major AGP in tomato, was examined in different organs and tissues of tomato (Lycopersicon esculentum Mill. cv. UC82B) plants with an anti-peptide antibody (i.e. the PAP antibody) directed specifically against the lysine-rich subdomain of the LeAGP-1 core protein. During cell differentiation in tomato plants, LeAGP-1 was associated with cell wall thickening and lignification of particular cell types. Specifically, LeAGP-1 was detected in secondary wall thickenings of maturing metaxylem and secondary xylem tracheary elements in roots and stems, and in thickened cell walls of phloem sieve elements. However, LeAGP-1 was also present in thin-walled, cortical parenchyma cells of seedling roots as well as thick-walled collenchyma cells in young stems, both of which are not lignified. Based on these observed patterns, possible roles for LeAGP-1 in plant growth and development are discussed. Received: 17 August 1999 / Accepted: 7 October 1999     

Minor vein structure and sugar transport in Arabidopsis thaliana

Leaf and minor vein structure were studied in Arabidopsis thaliana (L.) Heynh. to gain insight into the mechanism(s) of phloem loading. Vein density (length of veins per unit leaf area) is extremely low. Almost all veins are intimately associated with the mesophyll and are probably involved in loading. In transverse sections of veins there are, on average, two companion cells for each sieve element. Phloem parenchyma cells appear to be specialized for delivery of photoassimilate from the bundle sheath to sieve element-companion cell complexes: they make numerous contacts with the bundle sheath and with companion cells and they have transfer cell wall ingrowths where they are in contact with sieve elements. Plasmodesmatal frequencies are high at interfaces involving phloem parenchyma cells. The plasmodesmata between phloem parenchyma cells and companion cells are structurally distinct in that there are several branches on the phloem parenchyma cell side of the wall and only one branch on the companion cell side. Most of the translocated sugar in A. thaliana is sucrose, but raffinose is also transported. Based on structural evidence, the most likely route of sucrose transport is from bundle sheath to phloem parenchyma cells through plasmodesmata, followed by efflux into the apoplasm across wall ingrowths and carrier-mediated uptake into the sieve element-companion cell complex. Received: 5 October 1999 / Accepted: 20 November 1999     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999     

Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts

 When pumpkin (Cucurbita spp., cv. Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow No. 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium. These transgenic cells showed no morphological difference from wild-type cells. However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells. We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall. Received: 28 October 1999 / Accepted: 18 January 2000     

Influence of nitric oxide synthase activity on amino acid concentration in the quinolinate lesioned rat striatum: A microdialysis and histochemical study

Summary. The influence of nitric oxide synthase (NOS) activity on the KCl-evoked amino acid concentrations was investigated by in vivo microdialysis in the striatum in a rat model of excitotoxic lesion. Basal microdialysate levels of amino acids decreased during the quinolinic acid-induced neurodegeneration process, except for glutamine that increased initially and returned to control values 30 days after quinolinic acid exposure. KCl-evoked increase of extracellular amino acid concentration was reduced due to NOS activity in the striatum of both controls and lesioned animals, except for 120 days after quinolinic acid injection. These changes of amino acid concentrations in microdialysates correlated with the known biochemistry of the consecutive domineered cell types during the lesion process as revealed by histochemistry for NOS, NADPH-diaphorase, GFAP and isolectin B4. The present data provide direct evidence that NOS activity can modulate extracellular amino acid concentrations in the striatum not only under physiological conditions, but also during a pharmacologically induced lesion process and, thus, suggests that nitric oxide affects neurodegeneration via this pathway. Received October 20, 1999; Accepted February 25, 2000     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

A deterministic size-structured population model for the worm Naidis elinguis

In this paper we model the population dynamics of the worm Nais elinguis, which reproduces by division into two unequal parts. By using renewal theory we derive the asymptotic behaviour of a Naidis elinguis population. In particular we prove a certain relation between the fraction of the population that was born small (respectively the fraction that was born large) and the inter-division times. Received 20 January 1999 / Revised version: 1 August 1999?Published online: 10 April 2001     

Isolation and characterization of two acyl-CoA-binding proteins from proembryogenic masses of Digitalis lanata Ehrh.

 Two acyl-CoA-binding-protein (ACBP) isoforms were isolated from proembryogenic masses of Digitalis lanata Ehrh. by column chromatography and preparative HPLC. The ACBPs had molecular masses of 9926 and 9997 Da, respectively. Partial sequence data indicated high similarity to each other and to ACBPs of other plant species such as Ricinus communis, Brassica napus and Arabidopsis thaliana. The isolated ACBPs bound palmitoyl-CoA with high affinity as determined by isoelectric-point shift. Received: 29 May 1999 / Accepted: 28 August 1999     

Inhibition of ornithine decarboxylase by α-difluoromethylornithine induces apoptosis of HC11 mouse mammary epithelial cells

Summary. The effect of α-difluoromethylornithine (DFMO) on the apoptosis of HC11 mouse mammary epithelial cells was investigated. The involvement of reactive oxygen species (ROS) and Bcl-2 protein in the mechanism of apoptosis induced by ornithine decarboxylase (ODC) inhibition was also assessed. DFMO (0.1, 1 and 5 mM) induced apoptosis of HC11 cells in dose- and time-dependent manner. Apoptosis manifests itself with morphological features like: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis (putrosis). The decrease in the nuclear DNA contents appearing as the hypodiploidal peak sub-G₁ in the DNA histogram was not dependent on the presence of prolactin (5 μg/ml) in DFMO-treated cultures. Apoptosis induced by ODC inhibition was associated with a rapid increase in ROS concentration in HC11 cells observed within 1 h after DFMO treatment. The down-regulation of Bcl-2 as a decrease in cell number expressing bcl-2 and a lowered Bcl-2 protein content in cells expressing this protooncogene was also noted. The administration of putrescine (50 μM) lowered the number of early-apoptotic, late-apoptotic and necrotic cells. Moreover, it increased the number of cells expressing bcl-2. In conclusion, the disturbance of cellular polyamine homeostasis by inhibition of their synthesis enhances mammary epithelial cell susceptibility to apoptosis. It may occur in the mammary gland at the end of lactation, when the depletion of circulating lactogenic hormones and activation of intra-mammary apoptogenic factors expression take place. Received May 6, 1999; Accepted December 15, 1999     

Conversion of guaiacyl to syringyl moieties on the cinnamyl alcohol pathway during the biosynthesis of lignin in angiosperms

Aglycons derived from 4-O-β-D-glucosides of both caffeyl and 5-hydroxyconiferyl alcohols were incorporated into guaiacyl (G) and syringyl (S) units in the lignin of newly formed xylem of several angiosperms. It is likely that these aglycons enter the cinnamyl alcohol pathway as intermediates in the introduction of methoxyl groups onto aromatic rings, and serve as precursors for the biosynthesis of lignin. The S/G ratio in this pathway was coincident with the ratio in the cell wall lignin of each tree. Our results indicate that the cinnamyl alcohol pathway involves the same mechanisms as the cinnamic acid and cinnamyl CoA pathways and they suggest that this novel pathway might be part of a metabolic grid in the biosynthesis of lignin. Received: 8 September 1999 / Accepted: 4 October 1999     

The application of atomic force microscopy to topographical studies and force measurements on the secreted adhesive of the green alga Enteromorpha

Atomic force microscopy (AFM) enables the topographical structure of cells and biological materials to be resolved under natural (physiological) conditions, without fixation and dehydration artefacts associated with imaging methods in vacuo. It also provides a means of measuring interaction forces and the mechanical properties of biomaterials. In the present study, AFM has been applied for the first time to the study of the mechanical properties of a natural adhesive produced by a green plant cell. Swimming spores of the green alga Enteromorpha linza (L.) J. Ag. (7–10 μm) secrete an adhesive glycoprotein which provides firm anchorage to the substratum. Imaging of the adhesive in its hydrated state revealed a swollen gel-like pad, approximately 1 μm thick, surrounding the spore body. Force measurements revealed that freshly released adhesive has an adhesion strength of 173 ± 1.7 mN m⁻¹ (mean ± SE; n=90) with a maximum value for a single adhesion force curve of 458 mN m⁻¹. The adhesive had a compressibility (equivalent to Young's modulus) of 0.54 × 10⁶ ± 0.05 × 10⁶ N m⁻² (mean ± SE; n=30). Within minutes of release the adhesive underwent a progressive `curing' process with a 65% reduction in mean adhesive strength within an hour of settlement, which was also reflected in a reduction in the average length of the adhesive polymer strands (polymer extension) and a 10-fold increase in Young's modulus. Measurements on the spore surface itself revealed considerably lower adhesion-strength values but higher polymer-extension values than the adhesive pad, which may reflect the deposition of different polymers on this surface as a new cell wall is formed. The study demonstrates the value of AFM to the imaging of plant cells in the absence of fixation and dehydration artefacts and to the characterisation of the mechanical properties of plant glycoproteins that have potential utility as adhesives. Received: 22 February 2000 / Accepted: 20 April 2000     

Characterization of prosystemin expressed in the baculovirus/insect cell system reveals biological activity of the systemin precursor

 Tomato (Lycopersicon esculentum Mill.) prosystemin in fusion with a viral signal peptide was expressed in Sf21 insect cell cultures after infection with recombinant baculoviruses. Prosystemin was purified from culture supernatants and its identity was confirmed by N-terminal sequence and mass-spectral analyses. Recombinant prosystemin was found to be equally active as compared to systemin in inducing the expression of wound-response genes in tomato plants. In cultured cells of L. peruvianum, prosystemin elicited a rapid alkalinization of the growth medium. The timing and dose-dependence of the alkalinization response were found to be identical for prosystemin and systemin, respectively. Prosystemin-triggered defense responses were inhibited by a competitive antagonist of systemin activity, indicating that the systemin sequence within the primary structure of prosystemin determines its activity. Received: 30 August 1999 / Accepted: 6 December 1999     

The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans

The interaction between Phytophthora infestans (Mont.) de Bary and Solanum was examined cytologically using a diverse set of wild Solanum species and potato (S. tuberosum L.) cultivars with various levels of resistance to late blight. In wild Solanum species, in potato cultivars carrying known resistance (R) genes and in nonhosts the major defense reaction appeared to be the hypersensitive response (HR). In fully resistant Solanum species and nonhosts, the HR was fast and occurred within 22 h. This resulted in the death of one to three cells. In partially resistant clones, the HR was induced between 16 and 46 h, and resulted in HR lesions consisting of five or more dead cells, from which hyphae were occasionally able to escape to establish a biotrophic interaction. These results demonstrate the quantitative nature of the resistance to P. infestans. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones and correlated with resistance levels. Other responses associated with the defense reaction were deposition of callose and extracellular globules containing phenolic compounds. These globules were deposited near cells showing the HR, and may function in cell wall strengthening. Received: 22 April 1999 / Accepted: 4 November 1999     

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis

 A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53 094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in  E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4′-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments. Received: 8 September 1999 / Accepted: 4 October 1999     

The Golgi apparatus of the scaly green flagellate Scherffelia dubia: uncoupling of glycoprotein and polysaccharide synthesis during flagellar regeneration

The flagella of the green alga Scherffelia dubia are covered by scales which consist of acidic polysaccharides and glycoproteins. Experimental deflagellation results in the regeneration of flagella complete with scales. During flagellar regeneration, scales are newly synthesized in the Golgi apparatus, exocytosed and deposited on the growing flagella. Flagellar regeneration is dependent upon protein synthesis and N-glycosylation, as it is blocked by cycloheximide and partially inhibited by tunicamycin. Metabolic labeling with [³⁵S]methionine/cysteine demonstrated that scale-associated proteins were not newly synthesized during flagellar regeneration, suggesting that the proteins deposited on regenerating flagella were drawn from a pool. Quantitative immunoelectron microscopy using a monospecific antibody directed against a scale-associated protein of 126 kDa (SAP126) revealed that the pool of SAP126 was primarily located at the plasma membrane, with minor labeling of the scale reticulum and trans-Golgi cisternae, both before deflagellation and during flagellar regeneration. Since SAP126 was sequestered during flagellar regeneration into secretory vesicles together with newly synthesized scales, it is concluded that the persistent presence of SAP126 in the trans-Golgi cisternae during scale biogenesis requires retrograde transport of the protein from the plasma membrane to the Golgi apparatus. Received: 3 July 1999 / Accepted: 21 August 1999