Marina Ciancia

O-Glycosylated Cell Wall Proteins Are Essential in Root Hair Growth

Sequential protein posttranslational modifications facilitate cell wall self-assembly and root hair elongation in Arabidopsis. Root hairs are single cells that develop by tip growth and are specialized in the absorption of nutrients. Their cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins (HRGPs) that include extensins (EXTs) and arabinogalactan-proteins (AGPs). Proline hydroxylation, an early posttranslational modification of HRGPs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs (which are mainly arabinosylated) and AGPs (which are mainly arabinogalactosylated). We explored the biological function of P4Hs, arabinosyltransferases, and EXTs in root hair cell growth. Biochemical inhibition or genetic disruption resulted in the blockage of polarized growth in root hairs and reduced arabinosylation of EXTs. Our results demonstrate that correct O-glycosylation on EXTs is essential for cell-wall self-assembly and, hence, root hair elongation in Arabidopsis thaliana.

The Cell Walls of Green Algae: A Journey through Evolution and Diversity

The green algae represent a large group of morphologically diverse photosynthetic eukaryotes that occupy virtually every photic habitat on the planet. The extracellular coverings of green algae including cell walls are also diverse. A recent surge of research in green algal cell walls fueled by new emerging technologies has revealed new and critical insight concerning these coverings. For example, the late divergent taxa of the Charophycean green algae possess cell walls containing assemblages of polymers with notable similarity to the cellulose, pectins, hemicelluloses, arabinogalactan proteins (AGPs), extensin, and lignin present in embryophyte walls. Ulvophycean seaweeds have cell wall components whose most abundant fibrillar constituents may change from cellulose to β-mannans to β-xylans and during different life cycle phases. Likewise, these algae produce complex sulfated polysaccharides, AGPs, and extensin. Chlorophycean green algae produce a wide array of walls ranging from cellulose–pectin complexes to ones made of hydroxyproline-rich glycoproteins. Larger and more detailed surveys of the green algal taxa including incorporation of emerging genomic and transcriptomic data are required in order to more fully resolve evolutionary trends within the green algae and in relationship with higher plants as well as potential applications of wall components in the food and pharmaceutical industries.

Antiherpetic and anticoagulant properties of carrageenans from the red seaweed Gigartina skottsbergii and their cyclized derivatives: correlation between structure and biological activity

The antiviral activity against herpes simplex virus types 1 and 2 of kappa/l-, partially cyclized mu/v-, and lambda-carrageenans isolated from the red seaweed Gigartina skottsbergii and their cyclized derivatives was analyzed. lambda-Carrageenans and the partially cyclized mu/v-carrageenan were the most potent inhibitors of herpes viruses (including acyclovir-resistant variants and clinical isolates), with IC50 values lower than 1 microgram ml-1 against both serotypes and selectivity indices higher than 10(3). kappa/l-Carrageenans were slightly less effective than the other two types with IC50 values in the range 1.6-4.1 micrograms ml-1. Antiherpetic activity was directly correlated to the amount of alpha-D-galactose 2,6-disulfate residues in the natural carrageenans. The cyclization of the alpha-D-galactose 6-sulfate and 2,6-disulfate units into 3,6-anhydro-alpha-D-galactose and 3,6-anhydro-alpha-D-galactose 2-sulfate residues in these polysaccharides, in general, lowers the antiherpetic activity of the derivatives with respect to the natural carrageenans. Some carrageenans showed a very reduced anticoagulant activity only at concentrations that were considerably higher than the IC50, whereas others were totally devoid of anticoagulant properties. Among natural carrageenans, the mu/v-type IC3 shows the best relationship between antiviral efficacy and lack of anticoagulant action, resulting a very promising compound.

Antiherpetic activity and mode of action of natural carrageenans of diverse structural types

The lambda-carrageenan 1T1, the kappa/iota-carrageenan 1C1 and the mu/nu-type 1C3, isolated from the red seaweed Gigartina skottsbergii, proved to be potent and selective inhibitors of herpes simplex virus (HSV) types 1 and 2. The antiviral IC50 values determined by virus yield inhibition assay in different cell lines ranged from 0.4 to 3.3 microg/ml, and no cytotoxic effects, measured by trypan blue exclusion on stationary or proliferating cells, tetrazolium salt method or cell protein synthesis, were observed. Time of addition and attachment studies suggested that the main target for antiviral action of the three carrageenans was virus adsorption, whereas no effect on virus internalization, or early or late protein synthesis was detected. However, the lambda-carrageenan 1T1 was still significantly inhibitory when added any time after adsorption. The pretreatment of virions with the carrageenans showed that 1C1 and 1C3 lacked direct inactivating effect at concentrations near the antiviral IC50 but 1T1 exerted virucidal action. The cyclization of 1T1 to afford the derivative 1T1T1 maintained the antiviral activity but eliminated the virucidal properties. Thus, the structure of 1T1 seems to be responsible for its differential behavior from 1C1 and 1C3, probably allowing a more stable binding to HSV, leading to virion inactivation. In contrast, 1C1 and 1C3 fail to bind with high affinity to virus alone, but are able to interfere with the interaction between HSV particles and the cell.

Alkali-modification of carrageenans: mechanism and kinetics in the kappa/iota-, mu/nu- and lambda-series

Abstract The cyclization reaction (formation of 3,6-anhydro-α- d -galactose units from α- d -galactose 6-sulfate) of carrageenans follows a pseudo first-order kinetics, being 20–60 times faster for carrageenans of the kappa-family than for those of the lambda-family. In lambda-carrageenans the clustering of the sulfate groups around the hydroxyl on C-3 of the α-unit shields it from polarization or ionization, reducing the cyclization reaction rate. Furthermore, molecular models of lambda-carrageenans suggest an adequate geometry for the interaction between the hydroxyl group on C-3 of the α-unit and the sulfate group on C-2 of the β-unit. These observations also explain the lack of cyclized derivatives of lambda-carrageenans in nature. The ease with which the cyclization reaction occurs for carrageenans of the kappa-family indicates that the alkaline treatments used industrially could be carried out under milder conditions, giving products of high gel strength.

Chemical and in situ characterization of macromolecular components of the cell walls from the green seaweed Codium fragile.

A comprehensive analysis of the carbohydrate-containing macromolecules from the coencocytic green seaweed Codium fragile and their arrangement in the cell wall was carried out. Cell walls in this seaweed are highly complex structures composed of 31% (w/w) of linear (1-->4)-beta-D-mannans, 9% (w/w) of pyruvylated arabinogalactan sulfates (pAGS), and low amounts of hydroxyproline rich-glycoprotein epitopes (HRGP). In situ chemical imaging by synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy and by immunolabeling using antibodies against specific cell wall carbohydrate epitopes revealed that beta-d-mannans and pAGS are placed in the middle part of the cell wall, whereas HRGP epitopes (arabinogalactan proteins (AGPs) and extensins) are located on the wall boundaries, especially in the utricle apical zone. pAGS are sulfated at C-2 and/or C-4 of the 3-linked beta-L-arabinopyranose units and at C-4 and/or C-6 of the 3-linked beta-D-galactopyranose residues. In addition, high levels of ketals of pyruvic acid were found mainly at 3,4- of some terminal beta-D-Galp units forming a five-membered ring. Ramification was found at some C-6 of the 3-linked beta-D-Galp units. In agreement with the immunolabeled AGP epitopes, a nonsulfated branched furanosidic arabinan with 5-linked alpha-L-Araf, 3,5-linked alpha-L-Araf, and terminal alpha-L-Araf units and a nonsulfated galactan structure composed of 3-(3,6)-linked beta-D-Galp residues, both typical of type-II AG glycans were found, suggesting that AGP structures are present at low levels in the cell walls of this seaweed. Based on this study, it is starting to emerge that Codium has developed unique cell wall architecture, when compared, not only with that of vascular plants, but also with other related green seaweeds and algae.

Novel DL-Galactan Hybrids from the Red Seaweed Gymnogongrus Torulosus are Potent Inhibitors of Herpes Simplex Virus and Dengue Virus:

A novel series of DL-galactan hybrids extracted from the red seaweed Gymnogongrus torulosus, was evaluated for its in vitro antiviral properties against herpes simplex virus type 2 (HSV-2) and dengue virus 2 (DEN-2). These compounds were very active against both viruses with inhibitory concentration 50% (IC50) values in the range 0.6–16 μg/ml for HSV-2 and 0.19–1.7 μg/ml for DEN-2, respectively, as determined in a virus plaque reduction assay in Vero cells. The DL-galactans lacked of cytotoxic effects, on stationary as well as on actively dividing cells, and anticoagulant properties. Some of the compounds showed a variable level of direct inactivating effect on both virions, with virucidal concentration 50% values exceeding the IC50s obtained by plaque reduction assay. Full inhibitory activity was achieved when the galactans were present during virus adsorption period, suggesting that the mode of action of these compounds is an interference in the binding of the surface envelope glycoprotein with the cell receptor.

The system of low-molecular-weight carrageenans and agaroids from the room-temperature-extracted fraction of Kappaphycus alvarezii.

The room-temperature-extracted fraction from the red seaweed Kappaphycus alvarezii consists mainly of low-molecular-weight carrageenans, with structural dispersion around a basic kappa-pattern. This dispersion results from: (a) low percentages of 3,6-anhydrogalactose and the presence of precursor units; (b) important quantities of 6-O-methyl beta-D-galactose (4-sulfate) residues; (c) significant amounts of iota-repeating structure, and (d) small amounts of non-sulfated and disulfated beta-D-galactose residues. Significant quantities of alpha-L-galactose units suggest the presence of agaroids, as it has been reported in several other carrageenophytes.

Carrageenan systems from tetrasporic and cystocarpic stages of Gigartina skottsbergii

Abstract Cystocarpic and tetrasporangial plants of Gigartina skottsbergii produced different systems of carrageenans. These carrageenans were fractionated with potassium chloride and analysed. The ranges of precipitation, the molar ratios Gal:3,6-AnGal: sulphate and the IR spectra of the fractions indicated that the cystocarpic system was composed of similar amounts of gelling and soluble carrageenans of the K/1 - and μ/ν-type, respectively. The tetrasporic system was composed of a major fraction of the λ- type precipitated at 0.60–1.00 M KCl and two minor fractions, one of the λ-type precipitated at 1.10–1.30 M KCl and the other soluble in 2.00 M KCl with a λ- like IR spectrum but μ/ν- like analytical data (ω-carrageenan). The major cystocarpic and tetrasporic carrageenan fractions were submitted to alkaline treatment and further fractionation with potassium chloride. The modified cystocarpic polysaccharide fractions gave products comprising mainly gelling carrageenans while the modified tetrasporic polysaccharide fractions yielded soluble carrageenans in agreement with previously assigned structures.

Determination of the structures of cystocarpic carrageenans from Gigartina skottsbergii by methylation analysis and NMR spectroscopy

The combined use of methylation analysis and high-field 1H and 13C NMR spectroscopy allows the determination of the fine structure of the carrageenans produced by the cystocarpic stage of Gigartina skottsbergii.