Yariv Reagent Research Articles

Fractionation and characterization of cell wall polysaccharides from coffee (Coffea arabica L.) pulp

Cell wall polysaccharides were isolated by sequential extractions from coffee pulp, the main solid waste from coffee processing. Extractions were conducted with distilled water at room and boiling temperatures, 0.5 % ammonium oxalate and 0.05 M Na2CO3 to obtain pectic fractions. Hemicelluloses were extracted by using 2 M and 4 M NaOH. The composition of the hemicellulose fractions suggested the presence of xyloglucans, galactomannans and arabinogalactan-proteins (AGPs). The main part of the cell wall polysaccharides recovered from coffee pulp were pectins branched with arabinogalactans. Coffee pulp pectic fractions were low-methoxylated with various amounts of protein (0.5–8.4 %) and phenolics (0.7–8.5 %). Detection at 280 nm in the HPSEC analyses and radial gel diffusion assay using Yariv reagent indicated the presence of AGPs in most of these fractions. NMR analyses of chelating agent (CSP) and dialyzed water (WSPD) extracted pectins were carried out. The results demonstrated that CSP contains only AG I. On the other hand, AG I and AG II are present in WSPD, probably covalently linked to the pectic portion. Comparison with the literature indicated similarities between the cell wall polysaccharides from coffee pulp and green coffee beans.

A Functionalizable Analog of the Yariv Reagent for AGP Imaging using Fluorescence Microscopy.

Small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides have been instrumental in elucidating the localization and function of these glycans. Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death. There is currently no small molecule probe capable of fluorescently labeling AGPs. The Yariv reagents are the only small molecules that bind AGPs, and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex. However, the Yariv reagents are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy. A fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta. Herein, we describe the synthesis of an azido analog of the Yariv reagent that can be further functionalized with a fluorophore to provide a glycoconjugate that binds AGPs and is fluorescent. We show that the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the βGlcYariv reagent. Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.

Immunostimulating activities and anti-cancer efficacy of rhamnogalacturonan-I rich polysaccharide purified from Panax ginseng leaf

This study aimed to evaluate the anti-cancer effects as well as stimulation of various immune related factors such as complementary system, macrophages, and NK cells of polysaccharides isolated from Panax ginseng (P. ginseng) leaf. The polysaccharide fractions (PGEP, PGEP-I, –II, and –III) isolated from P. ginseng were contained rhamnose (Rha), arabinose (Ara), galactose (Gal), galacturonic acid (GalA), and glucuronic acid (GlcA), all of which are typical sugar components of rhamnogalacturonan (RG)-I. Only PGEP-I reacted with the β-glucosyl Yariv reagent (37.29%), indicating the presence of type-II arabinogalactan. Additionally, methylation analysis indicated the PGEP-I mainly comprised 19 different sugar linkages including 4-linked GalpA, 2,4-linked Rhap, 3,6-linked Galp, 5-linked Araf, terminal GlcpA, and terminal Arap which are characteristics of RG-I. Meanwhile, PGEP-I enhanced the production of cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor-α and augmented the complement system. The intravenous administration of PGEP-I activated the natural killer (NK) cells in a dose-dependent manner against YAC-1. Prophylactic intravenous administration of PGEP-I significantly suppressed Colon26-M3.1 carcinoma inoculation-induced lung cancer (48.7%). Moreover, the anti-cancer effects of PGEP-I were significant even though NK cell functions were blocked by anti-asialo GM1. The findings suggest that PGEP-I, an RG-I polysaccharide, not only activates the complement system, macrophages and NK cells, but also has strong anticancer effects that inhibit lung cancer.

Structural identification of active moiety in anti-tumor metastatic polysaccharide purified from fermented barley by sequential enzymatic hydrolysis

The potent anti-cancer metastatic effects induced by the immunostimulatory activities of intravenously administered BF–I, an active polysaccharide purified from fermented barley, were previously evaluated. This study aimed to evaluate the anti-cancer metastatic effect of orally administered BF–I and elucidated the microstructure and active moiety of BF–I using sequential enzymatic hydrolysis. In experiments using Colon26-M3.1 carcinoma, prophylactic oral administration of BF–I potently suppressed lung cancer metastasis. Furthermore, gas chromatography-mass spectrometry analysis of partially methylated alditol acetate showed that BF–I comprised 15 different glycosyl linkages, including 4-linked Xylp (16.1%), terminal Araf (12.4%), 4-linked Glcp (32.8%), 3-linked Glcp (13.5%), 3,6-linked Glcp (1.5%), and 3,6-linked Galp (5.3%). These glycosyl linkage combinations are typical for arabinoxylan, yeast-derived β-glucan, barley-derived β-glucan, and type II arabinogalactan. To clarify the microstructure, BF–I was sequentially degraded via linkage-specific enzymatic hydrolysis using five enzymes. Furthermore, the sugar composition, glycosyl linkage, and reactivity with the β-glucosyl Yariv reagent were analyzed, along with sequencing based on tandem mass spectra of partially degraded fragment fractions. The results confirmed that BF–I comprises a mixture of four different types of polysaccharides (arabinoxylan, yeast-derived β-glucan, barley-derived β-glucan, and type II arabinogalactan) with similar molecular weights. Furthermore, different polysaccharides contribute significantly to cytokine (interleukin-6, -12, and tumor necrosis factor-α) secretion by peritoneal macrophages. Therefore, the potent anti-cancer metastatic activity of BF–I is proposed to be related to the immunostimulatory activities of the four different types of polysaccharides.

Clarification of the structural features of Rhamnogalacturonan-I type polysaccharide purified from radish leaves

Determining the structure of REPI, an immunostimulatory polysaccharide fraction from radish leaves, is an important health objective. Herein, we show that REP-I contains nine different monosaccharides, including GalA (22.2%), Gal (32.6%), Ara (27.5%), and Rha (10.2%) as main sugars. REP-I was also reacted with β-glucosyl Yariv reagent (29.8%), suggesting the presence of the arabino-β-3,6-galactan. Furthermore, methylated-product analysis revealed that REP-I contains 13 different glycosyl linkages, including 4-linked GalpA (21.0%), 2,4-linked Rhap (7.0%), 4-linked Galp (5.8%), 5-linked Araf (10.1%), and 3,6-linked Galp (7.9%), which are characteristic of RG-I. Microstructural information was obtained by sequential degradation using four linkage-specific glycosylases and β-elimination, with fragments analyzed on the basis of sugar composition, methylation, and MS/MS spectra. The results show that the immunostimulatory activity of REP-I is possibly due to the structure of RG-I, which is composed of a main chain with repeating [→2)-Rhap-(1 → 4)-GalpA-(1→] linkage units and three side-chains: a branched α(1 → 5)arabinan, a β(1 → 4)galactan, and arabino-β-3,6-galactan, which are branched at the C(O)4 position of each Rha residue in the REP-I main chain.

Structural characterization of the carbohydrate and protein part of arabinogalactan protein from Basella alba stem and antiadhesive activity of polysaccharides from B. alba against Helicobacter pylori

BackgroundIncreasing drug resistance of Helicobacter pylori has highlighted the search for natural compounds with antiadhesive properties, interrupting the adhesion of H. pylori to stomach epithelia. Basella alba, a plant widely used in Asian traditional medicine, was investigated for its antiadhesive activity against H. pylori. MethodsB. alba extract FE was prepared by aqueous extraction. Polysaccharides were isolated from FE by ethanol precipitation and arabinogalactan-protein (AGP) was isolated with Yariv reagent. Carbohydrate analyses was performed by standard methods and sequence analysis of the protein part of AGP by LC-MS. In vitro adhesion assay of fluorescent-labelled H. pylori J99 to human AGS cells was performed by flow cytometric analysis. ResultsRaw polysaccharides (BA1) were isolated and 9% of BA1 were identified as AGP (53.1% neutral carbohydrates L-arabinose, D-galactose, rhamnose, 5.4% galacturonic acid, 41.5% protein). After deglycosylation of AGP, the protein part (two bands at 15 and 25 kDa in tricine SDS-PAGE) was shown to contain peptides like ribulose-bisphosphate-carboxylase-large-chain. Histological localization within the stem tissue of B. alba revealed that AGP was mainly located at the procambium ring. Functional assays indicated that neither FE nor BA1 had significant influence on viability of AGS cells or on H. pylori. FE inhibited the bacterial adhesion of H. pylori to AGS cells in a dose dependent manner. Best anti-adhesive effect of ~67% was observed with BA1 at 2 mg/mL. ConclusionThe data obtained from this study characterize in part the mucilage and isolated polysaccharides of B. alba. As the polysaccharides interact with the bacterial adhesion, a potential uses a supplemental antiadhesive entity against the recurrence of H. pylori after eradication therapy may be discussed.

The Chirality of Aggregated Yariv Reagents Correlates with Their AGP-Binding Ability.

Yariv reagents are glycosylated triphenylazo dyes that bind to arabinogalactan proteins (AGPs), proteoglycans found in plant cell walls that are integral for plant growth and development. Yariv reagents are widely utilized as imaging, purification, and quantification tools for AGPs and represent the only small molecule probe for interrogating AGP function. The ability of Yariv reagents to bind to AGPs is dependent on the structure of the terminal glycoside on the dye. The reason for this selectivity has not been understood until the present work. Using circular dichroism spectroscopy, we show that the Yariv reagents form supramolecular aggregates with helical chirality. More significantly, the ability of the Yariv reagent to bind AGPs is correlated with this helical chirality. This finding paves the way towards developing a more detailed understanding of the nature of the Yariv-AGP complex, and the design of AGP-binding reagents with higher affinities and selectivities.

Structural Basis of the Function of Yariv Reagent-An Important Tool to Study Arabinogalactan Proteins.

Arabinogalactan proteins are very abundant, heavily glycosylated plant cell wall proteins. They are intensively studied because of their crucial role in plant development as well as their function in plant defence. Research of these biomacromolecules is complicated by the lack of tools for their analysis and characterisation due to their extreme heterogeneity. One of the few available tools for detection, isolation, characterisation, and functional studies of arabinogalactan proteins is Yariv reagents. Yariv reagent is a synthetic aromatic glycoconjugate originally prepared as an antigen for immunization. Later, it was found that this compound can precipitate arabinogalactan proteins, namely, their ß-D-(1→3)-galactan structures. Even though this compound has been intensively used for decades, the structural basis of arabinogalactan protein precipitation by Yariv is not known. Multiple biophysical studies have been published, but none of them attempted to elucidate the three-dimensional structure of the Yariv-galactan complex. Here we use a series of molecular dynamics simulations of systems containing one or multiple molecules of ß-D-galactosyl Yariv reagent with or without oligo ß-D-(1→3)-galactan to predict the structure of the complex. According to our model of Yariv-galactan complexes, Yariv reagent forms stacked oligomers stabilized by π-π and CH/π interactions. These oligomers may contain irregularities. Galactan structures crosslink these Yariv oligomers. The results were compared with studies in literature.

Arabinogalactan-like Glycoproteins from Ulva lactuca (Chlorophyta) Show Unique Features Compared to Land Plants AGPs.

Arabinogalactan proteins (AGPs) encompass a diverse group of plant cell wall proteoglycans, which play an essential role in plant development, signaling, plant-microbe interactions, and many others. Although they are widely distributed throughout the plant kingdom and extensively studied, they remain largely unexplored in the lower plants, especially in seaweeds. Ulva species have high economic potential since various applications were previously described including bioremediation, biofuel production, and as a source of bioactive compounds. This article presents the first experimental confirmation of AGP-like glycoproteins in Ulva species and provides a simple extraction protocol of Ulva lactuca AGP-like glycoproteins, their partial characterization and unique comparison to scarcely described Solanum lycopersicum AGPs. The reactivity with primary anti-AGP antibodies as well as Yariv reagent showed a great variety between Ulva lactuca and Solanum lycopersicum AGP-like glycoproteins. While the amino acid analysis of the AGP-like glycoproteins purified by the β-d-glucosyl Yariv reagent showed a similarity between algal and land plant AGP-like glycoproteins, neutral saccharide analysis revealed unique glycosylation of the Ulva lactuca AGP-like glycoproteins. Surprisingly, arabinose and galactose were not the most prevalent monosaccharides and the most outstanding was the presence of 3-O-methyl-hexose, which has never been described in the AGPs. The exceptional structure of the Ulva lactuca AGP-like glycoproteins implies a specialized adaptation to the marine environment and might bring new insight into the evolution of the plant cell wall.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Investigations of changes in the arabinogalactan proteins (AGPs) structure, size and composition during the fruit ripening process

Arabinogalactan proteins (AGPs) are ubiquitous cell wall and plasma membrane components and are characterised by extensive glycosylation and heterogeneity of their carbohydrate and protein units. The aim of the study was to evaluate the structural features of AGPs present in apple fruits at different stages of the ripening process. AGPs were extracted using the Yariv reagent and examined using SDS-PAGE, immunoblotting, FT-IR, and AFM. In situ analysis, immunofluorescence (CLSM) and immunogold-labelling (TEM), were performed. We demonstrated that AGPs were indeed present in apple fruits at the different stages of the ripening process. The changes in the amount (1.52–2.08 mg g−1), diameter (152.73–75.05 nm), molecular mass (50–250 kDa), and distribution in the cell of AGPs demonstrate their variable presence and changeable structure during the ripening process. We propose specific wavenumbers, i.e. 1265 cm−1, 1117 cm−1, and 960 cm−1, which could be assigned to AGPs. The immunofluorescence and immunogold-labelling results indicate that the JIM13 antibody is the most characteristic for AGPs in apple fruits. This study quantitatively demonstrated for the first time that AGP accumulation occurs in ripe fruits, which is supported by the highest AGPs content, the highest molecular mass, and the appearance of a specific distribution pattern at the cellular level.

Contributions to Arabinogalactan Protein Analysis.

Arabinogalactan proteins (AGPs) are important plant proteoglycans involved in many development processes. In roots, AGPs occur in the cell wall of root cells and root cap-derived cells as well as in the secreted mucilage. Detection, localization , and quantification techniques are therefore essential to unravel the AGP diversity of structures and functions. This chapter details root-adapted immunocytochemical methods using monoclonal antibodies, and a collection of biochemical analysis protocols using β-D-glucosyl Yariv reagent for comprehensive AGP characterization.

Localization, Extraction, and Quantification of Plant and Algal Arabinogalactan Proteins.

Arabinogalactan proteins are a diverse group of cell wall-associated proteoglycans. While structural and molecular genetic analyses have contributed to the emerging improved understanding of the wide-range of biological processes in which AGPs are implicated; the ability to detect, localize, and quantify them is fundamentally important. This chapter describes three methods: histological staining, radial gel diffusion, and colorimetric quantification, each of which utilize the ability of Yariv reagent to bind to AGPs.

Immunocytochemical studies on the distribution of arabinogalactan proteins (AGPs) as a response to fungal infection in Malus x domestica fruit

Arabinogalactan proteins (AGPs) are cell components implicated in plant-microbe interactions. Despite the significance of AGPs in response to stress factors, their distribution during development of fungal disease in fruit is unknown. In our work, in situ analysis of AGP arrangement in fruit inoculated with Penicillium spinulosum during the consecutive days of infection development was carried out. For immunolocalization of AGPs, samples were incubated with JIM13, MAC207, LM2, and LM14 antibodies recognizing the AGP carbohydrate moieties. To analyse cell walls without proper action of AGP, an experiment with β-glucosyl Yariv reagent specifically binding AGPs was performed. The results showed an increase of signal fluorescence in the fruit after 16 days of fungal disease. Higher amounts of the examined epitopes were observed in the infection-altered sites of the fruit, in close vicinity to a surface filled by fungal spores. The results indicate that the Yariv reagent treatment induced progress of the fungal disease. Changes in the AGP presence during the fungal disease confirmed their involvement in defence against pathogen attack in fruit.

Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits

Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (Mr 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with β-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-Mr neutral AGP (SE-2; Mr approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked β-galactosyl backbone chains substituted with side chains of (1 → 6)-linked β-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-β-(1 → 3)-galactanase with the aid of endo-β-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-Mr fragments. These consisted of free L-Ara and Gal, and a series of β-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked β-galactosyl side chains of varying length along the (1 → 3)-linked β-galactosyl backbone chains.

Structural characteristics of a red ginseng acidic polysaccharide rhamnogalacturonan I with immunostimulating activity from red ginseng

BackgroundMany researchers reported that the various immune activities of red ginseng are due to acid polysaccharides. But, the exact structural characteristics of the acidic polysaccharide in red ginseng have not been fully elucidated. Therefore, we isolated the acidic polysaccharide from red ginseng and characterized the structural property of the active moiety of this polysaccharide, which contributes to the immunostimulatory activity of red ginseng. MethodsA polysaccharide (RGP-AP-I) was purified from red ginseng via size-exclusion chromatography using Sephadex G-100. Immunostimulatary activity of RGP-AP-I was investigated via anti-complementory and macrophage stimulatory activity. The structure of RGP-AP-I was characterized by HPLC, sugar composition, β-glucosyl Yariv reagent and methylation analysis. ResultsPeritoneal macrophages stimulated using RGP-AP-I significantly augmented the production of various cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-α. The primary structure of RGP-AP-I was elucidated by assessing its sugar composition and methylation analysis. RGP-AP-I is a 96 kDa acidic polysaccharide, and comprises nine different monosaccharides, which mainly include sugars such as rhamnose (Rha, 9.5%), galacturonic acid (GalA, 18.4%), galactose (Gal, 30.4%), and arabinose (Ara, 35.0%). RGP-AP-I exhibited an considerable reaction with the β–glucosyl Yariv reagent, revealing the presence of arabino-β-3,6-galactan. Methylation analysis indicated that RGP-AP-I comprises 21 different glycosyl linkages, such as 3-, 4-, 6- and 3,6-linked Galp; 5-linked Araf; 2,4-linked Rhap; and 4-linked GalAp, which are characteristics of rhamnogalacturonan I (RG-I). Conclusionwe assumed that the immunostimulatory activity of RGP-AP-I may be due to the RG-I structure, which comprises a main chain with a repeating linkage unit, [→2)-Rhap-(1→4)-GalAp-(1→] and three groups of side chains such as (1→5)-linked arabinan, (1→4)-linked galactan, and arabino-β-3,6-galactan, which branch at the C(O)4 positions of Rha residues in the main chain of RGP-AP-I.

Arabinogalactan Proteins and the Extracellular Matrix of Charophytes: A Sticky Business.

Charophytes represent the group of green algae whose ancestors invaded land and ultimately gave rise to land plants 450 million years ago. While Zygnematophyceae are believed to be the direct sister lineage to embryophytes, different members of this group (Penium, Spirogyra, Zygnema) and the advanced thallus forming Coleochaete as well as the sarcinoid basal streptophyte Chlorokybus were investigated concerning their vegetative extracellular matrix (ECM) properties. Many taxa exhibit adhesion phenomena that are critical for affixing to a substrate or keeping cells together in a thallus, however, there is a great variety in possible reactions to e.g., wounding. In this study an analysis of adhesion mechanisms revealed that arabinogalactan proteins (AGPs) are most likely key adhesion molecules. Through use of monoclonal antibodies (JIM13) or the Yariv reagent, AGPs were located in cell surface sheaths and cell walls that were parts of the adhesion focal zones on substrates including wound induced rhizoid formation. JIM5, detecting highly methyl-esterfied homoglacturonan and JIM8, an antibody detecting AGP glycan and LM6 detecting arabinans were also tested and a colocalization was found in several examples (e.g., Zygnema) suggesting an interplay between these components. AGPs have been described in this study to perform both, cell to cell adhesion in algae forming thalli and cell to surface adhesion in the filamentous forms. These findings enable a broader evolutionary understanding of the function of AGPs in charophyte green algae.

Analysis of AGP contribution to the dynamic assembly and mechanical properties of cell wall during pollen tube growth

Arabinogalactan proteins as cell wall structural proteins are involved in fundamental processes during plant development and growth. The aim of this study was to evaluate AGP function in the distribution of pectin, cellulose and callose along Fragaria x ananassa pollen tube and to associate the cell wall structure with local mechanical properties. We used Yariv reagent which interacts with AGPs and allows the observation of the assembly of cell walls without AGPs performing their function. Cytochemical, immunofluorescence labelling and atomic force microscope have been used to characterize the changes in cell wall structure and stiffness. It was shown that disordering of the structure of AGP present in cell walls affects the localization of cellulose, pectins and the secretion of callose. Changes in cell wall assembly are relevant to pollen tube mechanical properties. The stiffness gradient lengthwise through the axis of the pollen tube has demonstrated a significantly higher Young’s modulus of the shank region than the growth zone. It has been revealed that the apex of the pollen tube cultured in the presence of Yariv reagent is stiffer (1.68 MPa) than the corresponding region of the pollen tube grown under control conditions (0.13-0.27 MPa). AGP affects the structure of the cell wall by changing the distribution of other components and the modification of their localization, and hence it plays a significant role in the mechanical properties of the cell wall.

Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with β-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Polysaccharides from the South African medicinal plant Artemisia afra: Structure and activity studies

Artemisia afra (Jacq. Ex. Willd), is an indigenous plant in South Africa and other parts of the African continent, where it is used as traditional medicine mostly for respiratory conditions. The objective of this study was to investigate the structural features of the polysaccharides from the leaves of this plant, as well as the biological activities of the polysaccharide fractions against the complement assay. Leaves of Artemisia afra were extracted sequentially with organic solvents (dichloromethane and methanol), 50% aqueous ethanol, and water at 50 and 100°C respectively. The polysaccharide extracts were fractionated by ion exchange chromatography and the resulting fractions were tested for biological activity against the complement fixation assay. Active fractions were further fractionated using gel filtration. Monosaccharide compositions and linkage analyses were determined for the relevant fractions. Polysaccharides were shown to be of the pectin type, and largely contain arabinogalactan, rhamnogalacturonan and homogalacturonan structural features. The presence of arabinogalactan type II features as suggested by methylation analysis was further confirmed by the ready precipitation of the relevant polysaccharides with the Yariv reagent. An unusual feature of some of these polysaccharides was the presence of relatively high levels of xylose as one of its monosaccharide constituents. Purified polysaccharide fractions were shown to possess higher biological activity than the selected standard in the complement assay. Digestion of these polysaccharides with an endo-polygalacturonase enzyme resulted in polymers with lower molecular weights as expected, but still with biological activity which exceeded that of the standard. Thus on the basis of these studies it may be suggested that immunomodulating properties probably contribute significantly to the health-promoting effects of this medicinal plant.