Production of polysaccharides by Silene vulgaris callus culture depending on carbohydrates of the medium

Sources of carbohydrate nutrition such as sucrose, glucose, and galactose, with the exception of arabinose, were shown to influence positively callus growth and polysaccharide (pectin silenan and acidic arabinogalactan) biosynthesis. Galactose was found to cause a stimulatory effect on yield and productivity of arabinogalactan. Low concentrations of sucrose failed to support the cell growth and polysaccharide biosynthesis. Increasing sucrose concentrations led to biomass accumulation but failed to enhance efficiency of the substrate utilization. The optimal medium for the campion cell culture growth was found to be one containing 30 g/liter of sucrose or a mixture of sucrose with glucose (in 15 g/liter). Increasing sucrose concentrations in the medium from 30 to 100 g/liter failed to significantly influence the polysaccharide yields while the polysaccharide productivity per liter of the medium grew due to promotion of culture productivity in biomass. Variations of the carbon sources in the nutrient media were shown to influence insignificantly the biochemical characteristics of arabinogalactan and silenan while an increase in the sucrose concentration to 50-100 g/liter led to a diminution of the galacturonic acid content in silenan and to changes in contents of the neutral monosaccharide residues in silenan and arabinogalactan.     

Modification of polysaccharides from callus culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G. using carbohydrases <Emphasis Type="Italic">in vitro</Emphasis>

Polysaccharides (pectin and intracellular and extracellular arabinogalactans) were isolated from campion callus culture cultivated on medium with varied concentrations of pectinase and beta-galactosidase. A decrease in contents of arabinose residues in pectin and arabinogalactans and of galactose residues in arabinogalactans was associated with an increase in the activities of alpha-L-arabinofuranosidase and beta-galactosidase upon addition of pectinase into the medium. Pectinase destroyed the high-molecular-weight (more than 300 kD) fraction of pectin and decreased the content of galacturonic acid residues. alpha-L-Arabinofuranosidase transformed arabinogalactan into galactan, and galactan was destroyed under the influence of galactosidase. The contents of arabinogalactan and/or galactan in the cells were decreased, and it was released into the culture medium. Pectin samples with low contents of arabinose and galactose in the side chains and galactan samples were obtained from the callus grown on the medium with beta-galactosidase. Cultivation of the plant cells on medium containing carbohydrases resulted in modification of pectin and arabinogalactan of the cell walls.     

Polysaccharides of cell cultures of <Emphasis Type="Italic">Silene vulgaris</Emphasis>

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3–6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5–1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Pectin substances of the callus culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G.

Pectin-protein fraction SVC was isolated from the callus culture of the bladder campion (Silene vulgaris). The main components in it were residues of D-galacturonic acid, galactose, arabinose, rhamnose, and protein. Using ion-exchange chromatography, ultrafiltration, and acid and enzymatic hydrolysis, it was shown that SVC contained a mixture of molecules of linear pectin, branched pectin polysaccharide, and pectin-protein polymer. A fragment of the linear chain of galacturonan amounted to more than half of the entire carbohydrate silenan chain. The branched area of the macromolecule is represented by rhamnogalacturonan I. The pectin-protein polymer consisted mainly of protein and weakly branched pectin fragments with molecular mass of more than 300 kDa.     

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris

Ultraviolet radiation (wavelength, 280-315 nm; power, 0.2-13.0 W/m2; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m2) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1 : (3.4-8.3).     

An alternate carbon source for enhancing production of polysaccharides by Silene vulgaris callus

Pectin termed silenan and acidic arabinogalactan were isolated as cell-wall polysaccharides of Silene vulgaris callus in the presence of various carbon sources as components of the media. The maximum yields, productivity per litre of medium and production per day of acidic arabinogalactan, were achieved using glucose or galactose as the carbon source. Sucrose was found to increase the production of the polysaccharides. Yields, productivity and rate of production of arabinogalactan per day were decreased in the presence of arabinose. Yields of silenan, productivity and rate of production per day were closely related irrespective of the sugar used as the carbon source in the media (sucrose, glucose or galactose) and yields of silenan from the callus growing on arabinose were comparable. A concentration of sucrose in the 20-50 g/L range enhanced the biosynthesis of silenan and at 50 g/L the silenan contained the linear backbone and the ramified regions of the macromolecule.     

The effect of ultraviolet radiation on the growth and polysaccharide composition of a callus culture of Silene vulgaris

Ultraviolet radiation (wavelength, 280–315 nm; power, 0.2–13.0 W/m²; exposure, 1 or 3 h) was shown to change the growth of campion callus and the polysaccharide (pectin and arabinogalactan) composition of cell walls. An increase in the concentration of polysaccharides and a decrease in the content of arabinose and galactose residues in pectin and arabinogalactan were noted. For the majority of calluses, growth indices, specific growth rate, and biomass productivity (per 11 medium) were almost the same as in nonirradiated control cells. Maximum values of the growth index and specific growth rate, determined for dry biomass, were observed at a low dose of irradiation (0.2 W/m²) and an exposure of 3 h. A considerable decrease in the content of arabinose and galactose in pectin was noted at high doses of irradiation (exposure, 3 h). Samples of arabinogalactan were characterized by variable arabinose to galactose ratios, which were in the range 1: (3.4–8.3).     

Polysaccharides of cell cultures of Silene vulgaris

Callus and suspension cultures of campion (Silene vulgaris) produced pectin polysaccharides, similar in structure to the polysaccharides of intact plants. The major components of the pectins were D-galacturonic acid, galactose, arabinose, and rhamnose residues. The maximum content of pectins was found in callus. The monosaccharide composition of arabinogalactans isolated from cells and a culture medium of callus cultures were similar, with the ratio between arabinose and galactose of 1: (2.3-6.5) being retained. The arabinogalactans from the cells and culture medium of the suspension cultures also had a similar structure, and the arabinose to galactose ratio was 1: (1.5-1.8). In contrast to the callus cultures, the suspension cultures produced arabinogalactans with an increased content of arabinose residues and a decreased content of galactose residues. The greatest content of arabinogalactan was detected in the culture medium of the suspension cultures.     

Pectin substances of the callus culture of Silene vulgaris (M.) G

Pectin-protein fraction SVC was isolated from the callus culture of the bladder campion (Silene vulgaris). The main components in it were residues of D-galacturonic acid, galactose, arabinose, rhamnose, and protein. Using ion-exchange chromatography, ultrafiltration, and acid and enzymatic hydrolysis, it was shown that SVC contained a mixture of molecules of linear pectin, branched pectin polysaccharide, and pectin-protein polymer. A fragment of the linear chain of galacturonan amounted to more than half of the entire carbohydrate silenan chain. The branched area of the macromolecule is represented by rhamnogalacturonan I. The pectin-protein polymer consisted mainly of protein and weakly branched pectin fragments with molecular mass of more than 300 kDa.     

Isolation of Polysaccharides from the Callus Culture of Lemna minor L.

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minorL.) with water and ammonium oxalate. Residues of galactose and arabinose (ratio, (2.0–2.5) : 1) were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glycuronic acids, galactose, and arabinose. The percentages of arabinogalactan and pectin were similar. The yield of polysaccharide fractions did not depend on the method used for their isolation. Extraction with water, treatment of the biomass with aqueous formalin and dilute hydrochloric acid, and extraction with aqueous ammonium oxalate allowed us to obtain the pectin polysaccharide with the highest purity.     

Isolation of polysaccharides from callus culture of Lemna minor L

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minor L.) with water and ammonium oxalate. Residues of galactose and arabinose in the 2.0-2.5:1 ratio were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glucuronic acids, galactose, and arabinose. The percentage of arabinogalactan and pectin was similar. The yield of polysaccharide fractions did not depend on the method for their isolation. Extraction with water, treatment of the biomass with an aqueous solution of formalin and diluted hydrochloric acid, and extraction with an aqueous solution of ammonium oxalate allowed us to obtain the highest-purity pectin polysaccharide.     

An arabinogalactan protein isolated from medium of cell suspension cultures of Silybum marianum (L.)Gaernt

Cell suspension cultures of Silybum marianum secreted polymers extracellularly containing 97% carbohydrates and 3% proteins. Fractionation of polysaccharides by anion-exchange chromatography yielded an unbound neutral fraction composed of glucose, xylose, galactose, arabinose and rhamnose and a bound fraction in which galactose and arabinose were predominantly found. The bound fraction specifically bind to Yariv phenylglycoside suggesting the presence of an arabinogalactan protein (AGP). Further purification of the AGP was done by precipitation of the culture medium with the Yariv reagent. The precipitated AGP eluated as single peak by gel permeation with an average molecular weight of 100. Eighteen aminoacids were detected, Ser, Gly, Glu, Asp, Thr and Hyp being the major ones. Linkage analysis showed terminal and 1,3-linked arabinose and almost all galactose was present in the 1,3-galactopyranoside form. The NMR spectral data revealed residues of galactopyranose and arabinofuranose as constituents of AGP. This study is the first examination of an AGP secreted by S. marianum cells in suspension culture.     

Action of β-galactosidase in medium on the Lemna minor (L.) callus polysaccharides

The callus culture of duckweed cultivated on medium containing different concentrations of β-galactosidase was shown to produce the following polysaccharides: pectin lemnan LMC, intracellular AG1, and extracellular AG2 arabinogalactans. The samples of lemnan with 46% galactose residue reduction and 9-46% increased galacturonic acid residue content were obtained at β-galactosidase concentrations of 10⁻³-10⁻¹ mg/mL. The most substantial alterations in the sugar composition of pectin were found to occur in the fraction with a molecular mass of 100-300 kDa. Low concentrations of enzyme failed to influence the sugar composition of intracellular arabinogalactan, whereas high concentrations were shown to decrease the amount of arabinose residues in AG1 and to cause galactan formation. Extracellular galactan was found to be produced on the medium with 10⁻¹ and 1 mg/mL β-galactosidase whereas extracellular arabinogalactan AG2 was shown to be biosynthesized without β-galactosidase or at a β-galactosidase concentration of 10⁻³ mg/mL. Alterations in the sugar composition of polysaccharides were shown to be connected with the increasing activity of α-l-arabinofuranosidase and β-galactosidase, and with the decreasing activity of intracellular polygalacturonase.     

Influence of the fungus Trichoderma harzianum on the enzyme and polysaccharide composition of Silene vulgaris callus

Activities of polygalacturonase and 1,3-β-glucanase increased in campion (Silene vulgaris) callus cells during co-cultivation with the fungus Trichoderma harzianum. This was associated with a decrease in galacturonic acid residues in the pectic polysaccharide of campion silenan and also in the production of pectin by the callus. Co-cultivation of the callus and the fungus resulted in an increase in contents of arabinose residues in the intracellular arabinogalactan and in contents of galactose residues in the extracellular arabinogalactan.     

A high molecular arabinogalactan from Ribes nigrum L.: influence on cell physiology of human skin fibroblasts and keratinocytes and internalization into cells via endosomal transport

An arabinogalactan protein (F2) was isolated in 1.5% yield from the seeds of Ribes nigrum L. (Grossulariaceae) by aqueous extraction and a one-step anion exchange chromatography on DEAE-Sephacel with 24% galactose, 43% arabinose, and 20% xylose as main carbohydrate residues. Methylation analysis revealed the presence of a 1,3-/1,3,6-galactose backbone, side chains from arabinose in different linkages, and terminal xylose residues. The polysaccharide which turned out to be an arabinogalactan protein had a molecular weight of >10⁶ Da and deaggregated under chaotropic conditions. The cellular dehydrogenase activities (MTT and WST-1 tests) of human skin cells (fibroblasts, keratinocytes) as well as the proliferation rate of keratinocytes (BrdU incorporation ELISA) were significantly stimulated by the polymer at 10 and 100 μg/mL. F2 had no influence on differentiation status of keratinocytes and did not exhibit any cytotoxic potential (LDH test). The biological activity of F2 was not dependent on the high molecular weight. Influence of the polysaccharide on the gene expression of specific growth factors, growth factor receptors, signal proteins and marker proteins for skin cell proliferation, and differentiation by RT-PCR could not be shown. Gene array investigations indicated an increased expression of various genes encoding for catabolic enzymes, DNA repair, extracellular matrix proteins, and signal transduction factors. Removal of terminal arabinose residues by α-l-arabinofuranosidase did not influence the activity toward skin cells, while the treatment with β-d-galactosidase yielded an inactive polysaccharide. The FITC-labeled polysaccharide was incorporated in a time-dependent manner into human fibroblasts (laser scanning microscopy) via endosomal transport. This internalization of the polysaccharide was inhibited by Cytochalasin B.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Polysaccharides of Soybean Seeds

A polysaccharide fraction extracted from soybean seeds with boiling water was examined by several fractionation methods on ultracentrifugal criteria. Four components were found by a column chromatography using TEAE-cellulose or by a paper electrophoresis. Acetone-precipitation, fractionation by conversion into acetyl derivative, and copper-complex-precipitation were unsatisfactory to fractionate into the components. The major component (70%) isolated was an arabinogalactan containing residues of arabinose and galactose in the approximate proportion of 1 : 2 and having molecular weight of 3.3×10⁵.     

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, ¹³C-NMR, ¹H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.     

Stimulation of polysaccharide formation by 2,4-dichlorophenoxyacetic acid in callus tissues of Arabidopsis thaliana

A relatively high concentration of 2,4-dichlorophenoxyacetic acid (45 μ M ) in solid culture medium stimulated the formation and secretion of mucilage polysaccharides by callus tissues of Arabidopsis thaliana L. Heynh. (line Estland). The mucilage was composed of at least two polysaccharides as revealed by gel chromatography on Sepharose 4B: the major component (87%) eluted in the void volume (molecular weight 2 × 10⁶ or greater) and the minor component (13%) eluted in the molecular weight range from 2 × 10⁴ to 4 × 10⁵. Both polysaccharide components contained small amounts of uronic acids. The major polysaccharide consisted mostly of galactose (49%), arabinose (28%) and fucose (10%), whereas the minor one consisted of galactose (44%), xylose (18%), arabinose (14%) and rhamnose (14%). One of the components of the secreted mucilage seems to be an arabinogalactan.     

Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus

UV-C irradiation (254 nm) was found to enhance the secretion of some cell-wall-degrading enzymes, especially the following carbohydrases: beta-galactosidase, alpha-L-arabinofuranosidase, polygalacturonase, pectinesterase, cellulase, xylanase, and beta-xylosidase, in the campion callus, contributing thereby to an alteration in the polysaccharide structure. The relative amounts of the galactose and arabinose residues in pectin (silenan) and of arabinose in arabinogalactan of calli irradiated during the exponential phase were shown to decrease during the stationary phase. A decrease in the degree of SV methylesterification was found for the irradiated callus. These alterations were found to persist over a long period of culturing time. Decreasing the relative amounts of the arabinose residues in arabinogalactan and pectin and the galactose residues in silenan corresponded to increasing activity of alpha-L-arabinofuranosidase and beta-galactosidase, respectively, due to treatment with UV-C. UV-C irradiation may be used as a tool for modifying the structural features of the cell-wall polysaccharides, such as the relative amounts of galactose and arabinose residues in the side chains of polysaccharides, with the purpose of obtaining physiologically active polysaccharides with the desired properties and structural features.