Birgit Classen

Mucilage and polysaccharides in the halophyte plant species Kosteletzkya virginica: Localization and composition in relation to salt stress

Mucilage is thought to play a role in salinity tolerance in certain halophytic species by regulating water ascent and ion transport. The localization and composition of mucilage in the halophyte Kosteletzkya virginica was therefore investigated. Plants were grown in a hydroponic system in the presence or absence of 100mM NaCl and regularly harvested for growth parameter assessment and mucilage analysis with the gas liquid chromatography method. NaCl treatment stimulated shoot growth and biomass accumulation, had little effect on shoot and root water content, and reduced leaf water potential (Psi(w)), osmotic potential (Psi(s)) as well as stomatal conductance (g(s)). Mucilage increased in shoot, stems and roots in response to salt stress. Furthermore, changes were also observed in neutral monosaccharide components. Levels of rhamnose and uronic acid increased with salinity. Staining with a 0.5% alcian blue solution revealed the presence of mucopolyssacharides in xylem vessels and salt-induced mucilaginous precipitates on the leaf abaxial surface. Determination of ion concentrations showed that a significant increase of Na(+) and a decrease of K(+) and Ca(2+) simultaneously occurred in tissues and in mucilage under salt stress. Considering the high proportion of rhamnose and uronic acid in stem mucilage, we suggest that the pectic polysaccharide could be involved in Na(+) fixation, though only a minor fraction of accumulated sodium appeared to be firmly bound to mucilage.

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the β-glucosyl Yariv reagent

An arabinogalactan-protein (AGP) from pressed juice of Echinacea purpurea herb was isolated from a high molecular weight fraction by precipitation with the beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of other AGPs: i.e., a high amount of polysaccharide (83%) with a ratio of galactose to arabinose of 1.8:1, some uronic acids (4-5%), and a low protein content (7%) with high levels of serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.2 x 10(6) Da. Linkage and 13C NMR analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and terminal units of Araf-(1-->5)-Araf-(1-->. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule. Complete loss of reactivity toward the beta-glucosyl Yariv antigen was then noticed.

Structural investigations on arabinogalactan-protein from wheat, isolated with Yariv reagent.

For the first time a pure arabinogalactan-protein (AGP) could be isolated from whole grain of wheat ( Triticum aestivum L.) by performing a double precipitation with beta-glucosyl Yariv reagent. The putative bioactive AGP has been characterized with regard to its polysaccharide and protein parts. Analytical investigations by GLC-MS and (13)C NMR revealed a carbohydrate moiety consisting of a 1,3-Galp backbone, linked in position 6 to short 1,6-Galp-chains, terminating in Araf. In the protein part, a high content of hydroxyproline has been found, probably responsible for linkage between protein and polysaccharide moieties. The molecular mass of AGP has been determined by size exclusion chromatography with laser light scattering detection and found to be 125 kDa. Alkaline hydrolysis of the protein resulted in single carbohydrate moieties with a molecular mass of about 20 kDa, indicating that AGP from whole grain of wheat belongs to the wattle blossom type of AGPs.

Characterization and Immunolocalization of Arabinogalactan-Proteins in Roots of Echinacea purpurea

From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea L. Moench, arabinogalactan-proteins (AGPs), a class of proteoglycans proposed to be involved in cell differentiation and plant growth, were purified and characterized with regard to amino acid composition and structure of the polysaccharide moiety. The protein content of the AGP was 5.0 % (w/w) with the dominating amino acids Glx, Hyp, Asx, Ser, Thr and Ala. The highly branched polysaccharide moiety shows a linkage composition typical of AGPs with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Terminal units of glucuronopyranose acid were also detected. Furthermore, a new method for the localization of AGPs in plant tissue has been developed. The synthetic (beta- D-Glc)(3) Yariv phenylgycoside (betaGlcY) is known to specifically bind to AGPs. For immunolocalization, polyclonal betaGlcY-antibodies have been generated and were used to label Yariv-treated thin sections of roots from E. purpurea. After addition of the FITC-conjugated secondary antibody, the sections were analyzed by confocal laser scanning microscopy. AGPs are detected mainly in the central cylinder in the area of the xylem. Cell walls of vessels and tracheids are strongly labelled, especially at the inner area of the wall. Furthermore, there is intense labelling of the pit canals.

Antibodies against Yarivʼs Reagent for Immunolocalization of Arabinogalactan-Proteins in Aerial Parts of Echinacea purpurea

Arabinogalactan-proteins are glycoproteins that occur in higher plants and are involved in important processes like cell differentiation and plant growth. In the medicinal plant Echinacea purpurea L., they belong to the putative immunomodulating compounds and are structurally well characterized. For microscopic localization of arabinogalactan-proteins, synthetic (β-D-Glc)3 Yariv phenylglycoside that specifically binds to most plant arabinogalactan-proteins was used to label arabinogalactan-proteins in fresh cut sections of stems and petioles of Echinacea purpurea. Polyclonal antibodies against (β-D-Glc)3 Yariv phenylglycoside were used to detect the arabinogalactan-protein-(β-D-Glc)3 Yariv phenylglycoside complex. After addition of fluorescein isothiocyanate-conjugated secondary antibodies, the sections were analyzed by confocal laser scanning microscopy. Arabinogalactan-proteins are localized mainly in the central cylinder in the collateral vascular bundles, especially in the area of the xylem. In cell walls of fully differentiated vessels and tracheids, arabinogalactan-proteins have been detected mainly at the inner area of the wall close to the cell lumina. Intense labeling occurs around pit canals connecting adjacent vessels. Furthermore, arabinogalactan-proteins are present in the lumina of cells of the sclerenchyma caps and in companion cells of the phloem.

Evolution of plant cell wall: Arabinogalactan-proteins from three moss genera show structural differences compared to seed plants

Arabinogalactan-proteins (AGPs) are important proteoglycans of plant cell walls. They seem to be present in most, if not all seed plants, but their occurrence and structure in bryophytes is widely unknown and actually the focus of AGP research. With regard to evolution of plant cell wall, we isolated AGPs from the three mosses Sphagnum sp., Physcomitrella patens and Polytrichastrum formosum. The moss AGPs show structural characteristics common for AGPs of seed plants, but also unique features, especially 3-O-methyl-rhamnose (trivial name acofriose) as terminal monosaccharide not found in arabinogalactan-proteins of angiosperms and 1,2,3-linked galactose as branching point never found in arabinogalactan-proteins before.

Allergy-Protective Arabinogalactan Modulates Human Dendritic Cells via C-Type Lectins and Inhibition of NF-κB

Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study was to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3–grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif–containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs.

Reproduction of the Medicinal Plant Pelargonium sidoides via Somatic Embryogenesis

The medicinal plant Pelargonium sidoides DC. (Geraniaceae) was traditionally used for the treatment of the common cold and cough in South Africa. Today an aequous-ethanolic root extract from this plant is approved for the treatment of acute bronchitis and is globally marketed also as an immunostimulant. The increasing demand of the plant material for the industrial production indicates the need of new effective methods for the propagation of P. sidoides. Here we report somatic embryogenesis and in vitro plantlet regeneration from somatic cells of inflorescence shoots and petioles of P. sidoides. A one-week cultivation of explants in media containing different concentrations of thidiazuron (1, 2.2, 3, and 4 mg/L) followed by a cultivation period without phytohormones resulted in the induction of somatic embryos within 2-4 weeks. After 2-4 months, the embryos generated roots and could be transferred into a greenhouse, where flower formation took place and the development of seeds occurred with high germination rates. The root umckalin concentration, determined by high-performance thin-layer chromatography, was comparable to that of seed-cultivated plants (100 ± 6 vs. 113 ± 10 µg umckalin/g dried roots). For the first time, direct somatic embryogenesis has been established as an appropriate cultivation method for P. sidoides plants used as raw material in the pharmaceutical industry. Moreover, genetically identical plants (chemical races) can be easily generated by this procedure.

Influence of plant growth regulators on development and polysaccharide production of cell cultures of Pelargonium sidoides

Pelargonium sidoides is a traditional medicinal plant from South Africa. An aqueous-ethanolic formulation of the roots and tubers is approved for the treatment of acute bronchitis. Therefore propagation of the plant material by cell cultures and the extraction of potential pharmaceutical active compounds are of great interest. Calli were established on different media from roots and shoots of seedlings and softness and colour of the tissue were compared. Optimum growth of callus cultures was achieved in MS-medium containing 1 mg/L 2,4-D and 0.2 mg/L kinetin or 2.2 mg/L TDZ supplemented with 50 mg/L ascorbic acid and 50 mg/L citric acid. Accumulation of phenolic deposits, responsible for inhibition of growth was avoided by addition of ascorbic and citric acid and a short period of sub-culture. Furthermore, the influence of different phytohormones [2,4-D, kinetin, 6-benzylaminopurine (BAP), α -naphthaleneacetic acid (NAA), Thidiazuron (TDZ)] on the polysaccharide composition of the liquid media of suspension cultures was investigated. For the first time, arabinogalactan-proteins (AGPs) as bioactive components were isolated from cell cultures of P. sidoides. Keywords: Pelargonium sidoides , cell culture, arabinogalactan-protein, 2,4-dichlorophenoxyacetic acid, kinetin, β -glucosyl Yariv reagent. African Journal of Biotechnology , Vol 13(31) 3244-3251

Structural investigations on arabinogalactan-proteins from a lycophyte and different monilophytes (ferns) in the evolutionary context

Today, understanding of cell wall evolution is incomplete due to limited knowledge of cell wall structure of non-flowering plants. Besides polysaccharides, proteoglycans like arabinogalactan-proteins are important constituents of the cell wall of most if not all seed plants. This article provides the first detailed characterization of AGPs from a lycophyte and different ferns, which are the closest living relatives to seed plants. The amount of protein in fern AGPs (6-12%) was in a range comparable to most seed plant AGPs. However, it was higher for Lycopodium AGP (17%). The carbohydrate moieties of investigated AGPs showed typical features known for type-II arabinogalactans from seed plants and showed cross-reaction with polyclonal antibodies raised against Echinacea AGP. On the other hand, unusual structural components like high quantities of pyranosidic arabinose residues for Lycopodium AGP and 3-O-methyl-rhamnose for fern AGPs have been detected.