Polysaccharides from the callus tissue of the rowan tree Sorbus aucuparia L. stem

Polysaccharide fractions SAcI and SAcII were isolated from callus tissues of rowan tree stems. The SAcI fraction was shown to contain compounds belonging to the arabinogalactan II group. The SAcII fraction, called sorban, comprised pectic polysaccharides composing the protopectin complex of the cell wall callus. The SAcII fraction was found to contain a large amount of galacturonic acid residues and a set of neutral sugars characteristic of rhamnogalacturonan I. The composition and properties allowed a suggestion that the sorban backbone is mainly formed by 1,4-α-D-galactopyranosyluronic acid residues, while the neutral sugars are represented by 1,4-linked glucopyranose and xylopyranose residues, 1,5-linked arabinofuranose, 1,6-linked galactopyranose and mannofuranose residues as well as terminal glucopyranose and xylopyranose residues. The callus growth was shown to be associated with nearly a constant content of galacturonic acid and neutral sugar residues in sorban (fraction SAcII).     

Structural Studies on Pectin from Marsh Cinquefoil <Emphasis Type="Italic">Comarum palustre</Emphasis> L.

Pectin with [alpha]D(20) +192 degrees (c 0.1; water), named comaruman, was isolated from marsh cinquefoil Comarum palustre L., which is widespread in the European North. The sugar chain of comaruman contains residues of D-galacturonic acid (64%), D-galactose (13%), L-rhamnose (12%), L-arabinose (6%), and trace amounts of xylose and glucose. Partial acid hydrolysis and digestion with pectinase demonstrated that comaruman composed of the backbone comprised regions of linear alpha-1,4-D-galactopyranosyl uronan interconnected by numerous residues of alpha-1,2-L-rhamnopyranose. In addition to the backbone (core of the macromolecule), ramified regions are involved in comaruman and comprise alpha-2,4-L-rhamno-alpha-4-D-galacturonan with side chains consisting mainly of beta-1,4-linked residues of D-galactopyranose. The ramified region contains additionally residues of 5-O-substituted arabinofuranose and 3- and 6-O-substituted galactopyranose. The present 3,4- and 4,6-di-O-substituted residues of galactopyranose appear to be branching points of the side chains. Some galactopyranose residues were found to occupy the terminal positions of the side chains or appeared to be single sugar residues attached to the side chains. Methylation analysis data indicated that comaruman contains residues of terminal, 3- and 3,4-di-O-substituted galactopyranosyl uronic acid, which appeared to be constituents of the side chains, and the latter represented additionally branching points of the backbone.     

Variations in the structure of neutral sugar chains in the pectic polysaccharides of morphologically different carrot calli and correlations with the size of cell clusters

Carrot (Daucus carota L.) embryogenic callus (EC) loses its embryogenic competence and becomes nonembryogenic callus (NC) during long-term culture. With the loss of embryogenic competence, the cell clusters become smaller and the extent of intercellular attachments is reduced. Pectic fractions prepared from EC and NC were separated into two subfractions by gel filtration. A difference in sugar composition between EC and NC was found only in the high-molecular-mass (ca. 1300 kDa) subfraction, and the ratio of the amount of arabinose to that of galactose (Ara/Gal) was strongly and positively correlated with the size of cell clusters in several different cultures. From the results of sugar-composition and methylation analyses, and the results of treatment with exo-arabinanase, models of the neutral sugar chains of pectins from EC and NC are proposed. Both neutral sugar chains are composed of three regions. The basal region is composed of linearly linked arabinan 5-Ara_f> moieties in both types of callus. The middle galactan region is composed of 6-linked galactose, some of which branches at the 3 and 4 positions, and this region is larger and more frequently branched in NC than in EC. Finally, the terminal arabinan region is composed of 5-linked arabinose, branched at the 3 position, and the size of the terminal arabinan is larger in EC than in NC. The significance of the neutral sugar chains of pectins in the interaction of cell wall components and intercellular attachment is discussed.Abbreviations Ara/Gal ratio (w/w) of the amount of arabinose to that of galactose - EC embryogenic callus - NC non-embryogenic callus - T-Ara_f terminal arabinose The authors are grateful to Dr. Naoto Shibuya of the National Institute of Agrobiological Resources for his gift of exo-arabinanase.     

Analytical and structural features of the gum exudate from Combretum hartmannianum schweinf.

The gum exudate from Combretum hartmannianum is water-soluble, forms very viscous solutions, and contains galactose (22%), arabinose (43%), mannose (10%), xylose (6%), rhamnose (4%), glucuronic acid (6%), 4-O-methylglucuronic acid (2%), and galacturonic acid (7%). The acidic components produced on hydrolysis of the gum were 6-O-(β-D-glucopyranosyluronic acid)-D-galactose, and two saccharides that had the same chromatographic mobility, and contained mannose and galacturonic acid, and galactose and 4-O-methylglucuronic acid, respectively. Methylation and methanolysis of the gum indicated the presence of terminal uronic acid, rhamnose, xylose, galactose, arabinofuranose, and arabinopyranose. Controlled, acid hydrolysis indicated the presence of (1→3)-linked arabinopyranose side-chains and (1→6)-linked galactose residues. C. hartmannianum gum, when subjected to two Smith-degradations, yielded Polysaccharides I and II, both of which contained galactose, arabinose, and mannose. Insufficient crude gum was available for a complete structural study, but the molecule was shown to contain long, sparsely branched chains of (1→6)-linked galactose residues, to which are attached (1→3)-linked arabinose and (1→3)-linked mannose side-chains.     

Structural characterisation of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa

An anti-complementary arabinogalactan (AGIIb-1), isolated from the roots of Angelica acutiloba Kitagawa, has been subjected to methylation analysis, digestion with alpha-L-arabinofuranosidase, controlled Smith-degradation, and partial acid hydrolysis. AGIIb-1 consisted of arabinose, galactose, rhamnose, galacturonic acid, and glucuronic acid in the molar ratios 1.8-2.2:1.0:0.2-0.3:0.2-0.4:0.1. AGIIb-1 contained mainly an arabino-3,6-galactan moiety, and most of the Ara was present as alpha-L-arabinofuranosyl residues in the non-reducing terminals and the highly polymerised and branched side-chains which were attached mainly to positions 3 and 6 of (1----6)- and (1----3)-linked Gal, respectively. Some Ara-containing chains were also attached to (1----4)-linked Gal residues. The 13C-n.m.r. data for AGIIb-1 showed that the Galp was beta. Mild acid hydrolysis of AGIIb-1 yielded several linear and highly branched arabino-oligosaccharides, a neutral arabinogalactan, and two acidic arabinogalactans. Some arabino-oligosaccharides contained a (1----4)-linked Arap at the reducing terminal. The neutral arabinogalactan contained (1----3)-, (1----4)-, and (1----6)-linked and 3,6-di-O-substituted Gal, whereas the acidic arabinogalactans contained, in addition, non-reducing terminal GlcA, (1----4)-linked GalA, and 2,4-di-O-substituted Rha. The anti-complementary activity was decreased when AGIIb-1 was partially hydrolysed with mild acid (10mM HCl, 100 degrees, 10 min), but treatment with exo-alpha-L-arabinofuranosidase markedly enhanced the activity.     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.     

Structural study of bergenan,a pectin from <Emphasis Type="Italic">Bergenia crassifolia</Emphasis>

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains the residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of their aqueous solutions. The results of enzymatic hydrolysis by α-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear α-1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-I). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constitutent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the backbone by a 1,4-linkages and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked α-arabinofuranose, and 1,4- and 1,6-linked β-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Control of hemicellulose and pectin synthesis during differentiation of vascular tissue in bean (Phaseolus vulgaris) callus and in bean hypocotyl

Membrane fractions from bean hypocotyl or callus incorporate arabinose from UDP--L-arabinose into arabinan and xylose from UDP--D-xylose into xylan. The control of these syntheses has been studied during xylogenesis in stele and in xylogenesis induced in callus tissue. Induction of arabinan synthetase activity occurs during division and extension growth while that of xylan synthetase occurs subsequently during the period of secondary thickening of the cell wall. The xylan synthetase induction is correlated with the induction of phenylalanine ammonia-lyase and with lignin synthesis.Abbreviations PAL phenylalanine ammonia-lyase - NAA 3-naphthylacetic acid - CMD medium supplemented with 2,4-dichlorophenoxy-acetic acid and coconut milk - IM induction medium - MM maintenance medium - EDTA ethylendiamine tetracetate - TCA trichloroacetic acid - DEAE diethylaminoethyl - TLC thin layer chromatography - UDP uridine diphosphate     

Primary structure of the xylose-containingN-linked carbohydrate moiety from ascorbic acid oxidase ofCucurbita pepo medullosa

Ascorbic acid oxidase (E.C.1.10.3.3) from the green zucchini squash (Cucurbita pepo medullosa) is a copper-containing glycoprotein which catalyzes the reaction:l-ascorbic acid +1/2 O₂l-dehydroascorbic acid + H₂O. The carbohydrate content of the purified plant glycoprotein amounted to 3% (w/w), and monosaccharide analysis revealed the carbohydrate moiety to be of theN-glycosidic type. The carbohydrate chains were released from the apoenzyme by digestion with PNGase-F immobilized on Sepharose 4B. After fractionation on Bio-Gel P-2 and purification on Mono-Q, the neutral oligosaccharide was investigated by 500-MHz¹H-NMR spectroscopy. The primary structure of theN-linked carbohydrate chain was established to be: Abbreviations AAO ascorbic acid oxidase - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Man mannose - Xyl xylose - GLC gas-liquid chromatography - FPLC fast protein liquid chromatography - NMR nuclear magnetic resonance - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The pectic enzymes of Aspergillus niger. A mercury-activated exopolygalacturonase

1. An exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 290-fold purification and a recovery of 8·6%. 2. The enzyme displayed its full activity only in the presence of Hg²⁺ ions; K_A for mercuric chloride was about 6×10⁻⁸m. 3. The mercury-activated enzyme progressively removed the terminal galacturonic acid residues from α-(1→4)-linked galacturonide chains and converted digalacturonic acid, trigalacturonic acid, tetragalacturonic acid and pectic acid into galacturonic acid.     

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Effect of growth regulators concentrations on morphological development of meristem-tips in Dioscorea cayenensis-D. rotundata complex and D. praehensilis

The use of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-d) has played an important role in the production and maintenance of totipotent cereal callus. However, 2,4-d has been implicated in the loss of totipotency from barley callus. To examine the effect of 2,4-d on barley callus, regenerability and karyotype were examined over time as influenced by cultivar differences and 2,4-d levels, during a period in which initially vigorous plant regeneration typically declines dramatically. Higher (20.4–27.1 M) versus lower (6.8–13.6 M) concentrations of 2,4-d were positively associated with the number of green plantlets recovered from calli maintained for 10 and 16 weeks before transfer to regeneration media, and with the longevity of regenerability. There was a positive relationship between 2,4-d concentration and normal karyotype. We also investigated the use of phenylacetic acid for the initiation of regenerable barley callus. Very poor callus growth and plant regeneration was supported by phenylacetic acid.Abbreviations PAA phenylacetic acid - SPDL(s) single plant-derived lines(s) - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - MSO Murashige and Skoog medium lacking growth regulators     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

Structure of Plant Cell Walls : XII. Identification of Seven Differently Linked Glycosyl Residues Attached to O-4 of the 2,4-Linked l-Rhamnosyl Residues of Rhamnogalacturonan I

Seven differently linked glycosyl residues have been found to be glycosidically linked to O-4 of the branched 2,4-linked l-rhamnosyl residues contained in the rhamnosyl and galacturonosyl backbone of the cell wall pectic polysaccharide rhamnogalacturonan I. These seven glycosyl residues are, therefore, the first residues of at least seven different side chains attached to the rhamnogalacturonan backbone. These first side chain glycosyl residues are 5-linked l-arabinofuranosyl and terminal 3-, 4-, 6-, 2,6-, and 3,6-linked d-galactopyranosyl residues. The existence of at least seven different side chains in rhamnogalacturonan I indicates that rhamnogalacturonan I is either an exceedingly complex polysaccharide or that rhamnogalacturonan I is a family of polysaccharides with similar or identical rhamnogalacturonan backbones substituted with different side chains.     

A journey to the world of glycobiology

Finding of the deletion phenomenon of certain oligosaccharides in human milk and its correlation to the blood types of the donors opened a way to elucidate the biochemical basis of blood types in man. This success led to the idea of establishing reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins. N-Linked sugar chains were first released quantitatively as oligosaccharides by enzymatic and chemical means, and labelled by reduction with NaB³H₄. After fractionation, structures of the radioactive oligosaccharides were determined by a series of methods developed for the studies of milk oligosaccharides. By using such techniques, structural rules hidden in the N-linked sugar chains, and organ- and species-specific N-glycosylation of glycoproteins, which afforded a firm basis to the development of glycobiology, were elucidated. Finding of galactose deficiency in the N-linked sugar chains of serum lgG from patients with rheumatoid arthritis, and malignant alteration of N-glycosylation in various tumors opened a new research world called glycopathology.However, recent studies revealed that several structural exceptions occur in the sugar chains of particular glycoproteins. Finding of the occurrence of the Gal1-4Fuc1- group linked at the C-6 position of the proximal N-acetylglucosamine residue of the hybrid type sugar chains of octopus rhodopsin is one of such examples. This finding indicated that the fucosyl residue of the fucosylated trimannosyl core should no more be considered as a stop signal as has long been believed. Furthermore, recent studies on dystroglycan revealed that the sugar chains, which do not fall into the current classification of N- and O-linked sugar chains, are essential for the expression of the functional role of this glycoprotein.It was found that expression of many glycoproteins is altered by aging. Among the alterations of the glycoprotein patterns found in the brain nervous system, the most prominent evidence was found in P₀. This protein is produced in non-glycosylated form in the spinal cord of young mammals. However, it starts to be N-glycosylated in the spinal cord of aged animals.These evidences indicate that various unusual sugar chains occur as minor components in mammals, and play important roles in particular tissues.     

The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na₂CO₃ with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N,N-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4-linked galactans and 1,5-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.Abbreviations GLC gas-liquid chromatography - MS mass spectrometry - V₀ void volume - MW weight-average molecular weight - DMSO dimethylsulphoxide - EDTA ethylenediamine tetraacetic acid - TFA trifluoroacetic acid - CDTA N,N,N-tetraacetic acid     

Callus induction and plant regeneration from gladiolus

A method for the initiation of callus capable of plant regeneration from in vivo grown cormels of gladiolus (Gladiolus x grandiflorus Hort.) is described. Sliced cormels of the large-flowering hybrid, Peter Pears were cultured in vitro on a modified Murashige and Skoog medium, supplemented with various auxins. Yellow callus, which was either friable or compact, could be induced on all media tested. Callus induced on media with naphthaleneacetic acid failed to proliferate. Callus induced on media with 9 mM 2,4-dichlorophenoxyacetic acid showed the best growth. Addition of micro-elements and vitamins increased the induction and growth of callus capable of plant regeneration. Explants taken from the middle part of the cormels had the highest competence for callus initiation. Callus was induced on several gladiolus hybrids and the South African species G. garnierii Klatt. Callus induction was genotype dependent and among the cultivars tested, Peter Pears and White Prosperity were superior with respect to callus production on the media with either 2,4-dichlorophenoxyacetic acid or picloram. Plants were regenerated from yellow compact callus of all genotypes on media containing zeatin and benzyladenine in various concentrations.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog basal salt and vitamins (1962) - CI callus induction medium - NAA -naphthaleneacetic acid - BA 6-benzyladenine - picloram 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid - zeatin 6-[4-hydroxy-3-methylbut-2-enylamino]purine