Tolerance of Douglas Fir Somatic Plantlets to Aluminum Stress: Biological, Cytological, and Mineral Studies.

Aluminum (Al) is well known as a potent inhibitor of plant growth and development. It is notably present in soils in the soluble and bioavailable form Al3+ when the soil pH drops below 5. This situation is frequent, especially in softwood forests when litter decomposition is slow. In the present work, we studied the effects of Al3+ on the growth and development of Douglas fir plantlets. Somatic plantlets, regenerated via somatic embryogenesis, were grown in vitro on media supplemented with different concentrations of aluminum chloride (AlCl3): 0 µM, 200 µM, 500 µM. and 1 mM. We show that a concentration of 500 µM AlCl3 in medium significantly reduced root elongation (-21.8%), as well as stem growth (-14.6%). Also, a 25% reduction in dry mass of the plantlets was observed in presence of a concentration of 200 µM of AlCl3. Histological analysis of root tissues revealed significant damage, especially in conducting vessels. In addition, mineral cation content of plantlets was disturbed under Al exposure. More particularly, the Mg and K contents of needles and the Ca content of stems and needles were significantly reduced in presence of a concentration of 500 µM AlCl3 in the culture medium (-35.6%, -33.5%, -24%, and -34% respectively). However, all these damages appeared at relatively high Al concentrations when compared with other herbaceous species. This study shed light on the ability of Douglas fir in vitro plantlets to cope with the acid-driven toxicity of Al.

Use of a Pleurotus ostreatus Complex Cell Wall Extract as Elicitor of Plant Defenses: From Greenhouse to Field Trial.

Fungi constitute an abundant source of natural polysaccharides, some of them harboring original structures which can induce responses in mammalian or plant cells. An alkaline extract from the edible mushroom Pleurotus ostreatus has been obtained and called Pleuran complex cell wall extract (CCWE). It consists of a glucan-peptide complex whose components fall in a quite broad range of molecular weights, from 30 to 80 kDa. Pleuran extract has been tested on cultivated plants in laboratory conditions and also during field trial for its capacity to stimulate plant defenses in response to pathogen attack. Following Pleuran CCWE treatment, enhanced levels of various biochemical markers associated with plant responses have been observed, including enzymatic activities (e.g., peroxidase) or expression of some pathogenesis-related genes. In addition, during field experiments, we have noticed significant reductions in disease symptom levels in relation to different plant/pathogen systems (wheat/septoria, vine/mildew). These results confirmed that Pleuran CCWE could be used as an elicitor of plant defenses and could help in reducing pesticide applications against plant pathogens.

Fractal structures and silica films formed by the Treignac water on inert and biological surfaces.

The Treignac water is a natural mineral water containing mainly orthosilicic acid. On inert substrates, it forms a silica film with fractal structures which cannot be reproduced in laboratory-reconstituted water. These structures form by condensation of orthosilicic acid monomers, following the Witten-Sander model of diffusion-limited aggregation. On biological surfaces, such as tomato leaves, the Treignac water forms a silica film with a different morphology and devoid of fractal structures. The filmogenic properties of this natural mineral water are here discussed in the context of crop protection, as the silica film can provide a barrier and a platform for the immobilization of elicitors of plant defense responses.

Antibacterial activity of the lichens Usnea Florida and Flavoparmelia caperata (Parmeliaceae).

Acetone extracts of the two common epiphytes lichens Usnea florida and Flavoparmelia caperata have been evaluated for their antimicrobial activities against Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis. The dibenzofuran derivative (+)-usnic acid (1) was the main metabolite in these two species. Thamnolic (5), evernic (6), physodic (7) and 3-hydroxyphysodic acids (8) were isolated from U. florida, as well as 5,7-dihydroxy-6-methylphtalide (2) which was newly identified in this Genus. Protocetraric (3) and caperatic acids (4) and ergosterol peroxide (9) are usually biosynthezised by F. caperata. Antibacterial activity was determined for the four main compounds against Staphylococcus aureus using bioautography and broth dilution method. Minimal inhibitory concentrations of usnic acid, caperatic acid and protocetraric acid were comprised between 7.25 and 12.5 µg/mL.

Agar Extraction By-Products from Gelidium sesquipedale as a Source of Glycerol-Galactosides.

Alkaline treatment is a common step largely used in the industrial extraction of agar, a phycocolloid obtained from red algae such as Gelidium sesquipedale. The subsequent residue constitutes a poorly valorized by-product. The present study aimed to identify low-molecular-weight compounds in this alkaline waste. A fractionation process was designed in order to obtain the oligosaccharidic fraction from which several glycerol-galactosides were isolated. A combination of electrospray ion (ESI)-mass spectrometry, ¹H-NMR spectroscopy, and glycosidic linkage analyses by GC-MS allowed the identification of floridoside, corresponding to Gal-glycerol, along with oligogalactosides, i.e., (Gal)2⁻4-glycerol, among which α-d-galactopyranosyl-(1→3)-ß-d-galactopyranosylα1-2⁻glycerol and α-d-galactopyranosyl-(1→4)-ß-d-galactopyranosylα1-2⁻glycerol were described for the first time in red algae.

Structural Investigation of Cell Wall Xylan Polysaccharides from the Leaves of Algerian Argania spinosa.

Xylan-type polysaccharides were isolated from the leaves of Argania spinosa (L.) Skeels collected in the Tindouf area (southwestern Algeria). Xylan fractions were obtained by sequential alkaline extractions and purified on Sepharose CL-4B. The xylan structure was investigated by enzymatic hydrolysis with an endo-ß(1→4)-xylanase followed by chromatography of the resulting fragments on Biogel P2, characterization by sugar analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS ). The results show that the A. spinosa xylan is composed of a ß-(1→4)-d-xylopyranose backbone substituted with 4-O-methyl-d-glucuronic acid and L-arabinose residues.

Physiological responses of the hybrid larch (Larix × eurolepis Henry) to cadmium exposure and distribution of cadmium in plantlets.

Phytoextraction of Cd is a growing biotechnology although we currently know few Cd hyperaccumulators, i.e., plant species able to accumulate at least 0.1 mg Cd g(-1) dry weight in aerial organs. Owing their deep root system and high biomass, trees are more and more preferred to herbaceous species for phytoextraction. Assuming that conifers could be relevant models under cold climates, we investigated cadmium tolerance of the hybrid larch Larix × eurolepis Henry (Larix decidua × Larix kaempferi) and the efficiency of this species to store this metal. In vitro grown larches were chosen in order to reduce time of exposure and to more rapidly evaluate their potential efficiency to accumulate Cd. One-month-old plantlets were exposed for 2 and 4 weeks to 250 and 500 µM Cd. Results showed that they tolerated a 4-week exposure to 250 µM Cd, whereas the content of photosynthetic pigment strongly dropped in plantlets growing in the presence of 500 µM Cd. In the presence of 250 µM Cd, shoot growth slightly decreased but photosynthetic pigment and total soluble carbohydrate contents were not modified and no lipid peroxidation was detected. In addition, these plantlets accumulated proline, particularly in shoots (two to three times more than control). In roots, Cd concentration in the intracellular fraction was always higher than in the cell wall fraction contrary to shoots where Cd concentration in the cell wall fraction increased with time and Cd concentration in the medium. In shoots, Cd concentration was lower than in roots with a ratio of 0.2 after 4 weeks of exposure but stayed around 0.2 mg g(-1) dry weight, thus a value higher than the threshold requested for Cd hyperaccumulators. Hybrid larch would thus be a relevant candidate for field test of Cd phytoextraction.

Douglas fir (pseudotsuga menziesii) plantlets responses to as, PB, and sb-contaminated soils from former mines.

Phytoremediation of metalloids by conifers is not widely studied although they may be relevant for several contaminated sites, especially those located in cold areas and sometimes under dry climates. Here, seeds of Douglas fir were sown in greenhouse on three soils collected in two French former mines: a gold mine (soils L1 and L2) and a lead and silver mine (soil P). These soils are highly contaminated by Pb, As, and Sb at different concentrations. Plants were harvested after ten weeks. Growth parameters, primary metabolite content, and shoot and root ionomes were determined. Douglas firs grown on the soils L1 and P had a lower biomass than controls and a higher oxidation status whereas those grown on the soil L2 exhibited a more developed root system and only slight modifications of carbon and nitrogen nutrition. Based on trace element (TE) concentrations in shoots and roots and their translocation factor (TF), Douglas fir could be a relevant candidate for As phytoextraction (0.8 g. kg(-1) dry weight in shoots and a TF of 1.1) and may be used to phytostabilize Pb and Sb (8.8 g and 127 mg. kg(-1) in roots for Pb and Sb, respectively, and TF lower than 0.1).

Interfacial reactions between humic-like substances and lateritic clay: application to the preparation of "geomimetic" materials.

The aim of this study was to understand the mechanisms responsible for the strengthening of "geomimetic" materials, especially the chemical bonding between clay and humic substances. The mineral matter is lateritic clay which mainly consists in kaolinite, goethite, hematite and quartz. The other starting products are fulvic acid (FA) and lime. The preparation of these geomimetic materials is inspired from the natural stabilization of soils by humic substances occurring over thousands of years. The present process involves acidic and alkaline reactions followed by a curing period of 18days at 60°C under a water saturated atmosphere. The acceleration of the strengthening process usually observed in soils makes this an original process for treatment of soils. The consolidation of the "geomimetic" materials could result from two major phenomena: (i) chemical bonding at the interface between the clay particles and iron compounds and the functional groups of the fulvic acid, (ii) a partial dissolution of the clay grains followed by the precipitation of the cementitious phases, namely calcium silicate hydrates, calcium aluminate hydrates and mixed calcium silicum and aluminum hydrates. Indeed, the decrease of the BET specific area of the lateritic clay after 24 h of reaction with FA added to the structural reorganization observed between 900 and 1000°C in the "geomimetic" material, and to the results of adsorption measurements, confirm the formation of organo-ferric complexes. The presence of iron oxides in clay, in the form of goethite, appears to be another parameter in favor of a ligand exchange process and the creation of binding bridges between FA and the mineral matter. Indeed all faces of goethite are likely to be involved in complexation reactions whereas in lateritic clay only lateral faces could be involved. The results of the adsorption experiments realized at a local scale will improve our understandings about the process of adsorption of FA on lateritic clays and its involvement in the strengthening process of materials.

Uncommon chlorinated xanthone and other antibacterial compounds from the lichen Cladonia incrassata.

Bioassay-guided fractionation of an extract of the lichen Cladonia incrassata against Staphylococcus aureus led to a novel compound, 1,5-dihydroxy-2,4,6-trichloro-7-methylxanthone (1), along with six known compounds: (-)-usnic acid (2), didymic acid (3), condidymic acid (4), squamatic acid (5), thamnolic acid (6), and prasinic acid (7). Didymic, condidymic, and prasinic acids were isolated for the first time from C. incrassata. Didymic, condidymic, and (-)-usnic acids were active against S. aureus (a minimum inhibitory concentration of 7.5 µg/mL).

Partial structural characterization and antioxidant activity of a phenolic-xylan from Castanea sativa hardwood.

4-O-Methylglucuronoxylans (MGX) were isolated from chestnut wood sawdust using two different procedures: chlorite delignification followed by the classical alkaline extraction step, and an unusual green chemistry process of delignification using phthalocyanine/H2O2 followed by a simple extraction with hot water. Antioxidant properties of both MGX were evaluated against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) by electronic spin resonance (ESR). IC50 of water-extracted MGX was found to be less than 225 µg mL(-1), in contrast with alkali-extracted MGX for which no radical scavenging was observed. Characterization of extracts by colorimetric assay, GC, LC-MS and NMR spectroscopy provided some clues to understanding structure-function relationships of MGX in connection with their antioxidant activity.

Phytoremediation of cadmium-contaminated soils by young Douglas fir trees: effects of cadmium exposure on cell wall composition.

Douglas fir trees grown on an artificially Cd-contaminated soil, can tolerate this trace element (up to 68 mg/kg in soil) during several months. Most of the absorbed Cd is retained in roots (25 mg/kg DM), but transfer to aerial part is also effective. Showing the highest content, up to 6 mg/kg DM, among all the aboveground parts, barks seem to be a preferred storage compartment. However, the transfer factor is quite low, about 0.3. Another objective of this study was to compare the cell wall components of trees exposed to increasing Cd amounts in soil. A decrease in lignin and an increase in pectin contents were observed in response to increasing soil cadmium concentration. A concurrent reduction in methyl-esterification of pectin suggests than the structure of this major binding site could therefore be modified as a reaction to cadmium contamination. Future prospects will focus on the modulation of pectin composition in response to Cd exposure.

Optimization of the arsenazo-III method for the determination of uranium in water and plant samples.

This work reports a reliable, fast and optimized photometric technique based on the specific chemical complexation of uranyl ion with arsenazo-III. In the case of solid samples (plant samples), for which mineralization under acidic and oxidative conditions was used, addition of ascorbic acid led to stabilization of the arsenazo-uranyl complex over time. The results, in total agreement with data obtained from α and γ spectrometries, demonstrate that the present technique is able to precisely quantify uranium in water as well as in plant samples, within the µg/L and mg/g ranges respectively.

Hybrid larch (Larix x eurolepis Henry): a good candidate for cadmium phytoremediation?

Studies related to phytoremediation by conifers are still at their beginning. Thus, we investigated the ability of a hybrid larch (Larix x eurolepis) to accumulate cadmium (Cd). One-month-old clonal plantlets grown in vitro were exposed for 1 week to a high Cd concentration (1.5 mM). No significant effect was observed on root and shoot biomass, root length, and needle number as a result of Cd treatment. Leaf photosynthetic pigment content and total soluble protein concentration in roots and shoots remained unchanged compared to control plantlets. Taken together, these results suggested that hybrid larch tolerated Cd in our conditions. The high Cd concentration in shoots (200 µg Cd gram(-1) dry weight) showed the good capacity of larch to translocate Cd and thus a potential use of this species for phytoremediation. Furthermore, under our conditions, phytochelatin biosynthesis pathway was slightly stimulated, suggesting that this pathway did not reach the threshold and/or another mechanism of Cd storage may be involved to explain larch tolerance to Cd.

Structural characterization and cytotoxic properties of an apiose-rich pectic polysaccharide obtained from the cell wall of the marine phanerogam Zostera marina.

Zosterin, an apiose-rich pectic polysaccharide, was extracted and purified from the sea grass Zostera marina. Structural studies conducted by gas chromatography and NMR spectroscopy on a purified zosterin fraction (AGU) revealed a typical apiogalacturonan structure comprising an alpha-1,4-d-galactopyranosyluronan backbone substituted by 1,2-linked apiofuranose oligosaccharides and single apiose residues. The average molecular mass of AGU was estimated to be about 4100 Da with a low polydispersity. AGU inhibited proliferation of A431 human epidermoid carcinoma cells with an approximate IC(50) value of 3 microg/mL (0.7 microM). In addition, AGU inhibited A431 cell migration and invasion. Preliminary experiments showed that inhibition of metalloproteases expression could play a role in these antimigration and anti-invasive properties. Autohydrolysis of AGU, which eliminated apiose and oligo-apiose substituents, led to a virtual disappearance of cytotoxic properties, thus suggesting a direct structure-function relationship with the apiose-rich hairy region of AGU.

Aqueous extraction of glucuronoxylans from chestnut wood: new strategy for lignin oxidation using phthalocyanine or porphyrin/H2O2 system.

Hardwood 4-O-methylglucuronoxylans (MGX) are classically isolated by a delignification step, using a sodium chlorite solution, followed by alkaline extraction, an efficient, although potentially polluting process. In this work, we propose a chlorine-free environmentally-friendly process for MGX extraction from Castanea sativa Mill. Chestnut sawdust was first delignified using metalled phthalocyanine or porphyrin in presence of hydrogen peroxide. Then, MGX were easily extracted by hot water. This protocol, repeated with different incubation times and temperatures, led to the selective extraction of acetylated MGX with decent yields reaching 12%. The best results were obtained after delignification using iron tetrapyrrole macrocycles. However, lower degrees of polymerization of MGX were observed in comparison to the classical method, suggesting that this new process affects the structures of xylans.

Structural characterization and cytotoxic properties of a 4-O-methylglucuronoxylan from castanea sativa. 2. Evidence of a structure-activity relationship.

Xylans were purified from delignified holocellulose alkaline extracts of Castanea sativa (Spanish chestnut) and Argania spinosa (Argan tree) and their structures analyzed by means of GC of their per-trimethylsilylated methylglycoside derivatives and (1)H NMR spectroscopy. The structures deduced were characteristic of a 4-O-methylglucuronoxylan (MGX) and a homoxylan (HX), respectively, with degrees of polymerization ranging from 182 to 360. In the case of MGX, the regular or random distribution of 4-O-methylglucuronic acid along the xylosyl backbone--determined by MALDI mass spectrometry after autohydrolysis of the polysaccharide--varied and depended both on the botanical source from which they were extracted and on the xylan extraction procedure. The MGX also inhibited in different ways the proliferation as well as the migration and invasion capability of A431 human epidermoid carcinoma cells. These biological properties could be correlated with structural features including values of the degree of polymerization, 4-O-MeGlcA to xylose ratios, and distribution of 4-O-MeGlcA along the xylosyl backbone, giving evidence of a defined structure-activity relationship.

Structural patterns of rhamnogalacturonans modulating Hsp-27 expression in cultured human keratinocytes.

Polysaccharide extracts were obtained from chestnut bran (Castanea sativa), grape marc (Vitis vinifera) and apple marc (Malus spp.) and fractionated by size exclusion chromatography after endopolygalacturonase degradation. Compositional and linkage analyses by GC and GC-MS showed the characteristic rhamnogalacturonan structure with specific arabinan (apple marc) and type II arabinogalactan (chestnut bran, grape marc) side chains. Type II arabinogalactan rhamnogalacturonan from chestnut bran significantly stimulated the in vitro differentiation of human keratinocytes, giving evidence of a tight structure-function relationship. This molecule comprises short and ramified 3- and 3,6-beta- D-galactan and 5- and 3,5-alpha-L-arabinan side chains, but also contains significant amounts of t-Xyl and 4-Xyl with a characteristic 2:1 ratio. Enzymatic hydrolysis of this polysaccharide produced fragments of lower molecular weight with unchanged xylose content which conserved the same ability to stimulate human keratinocyte differentiation. It could be then speculated that dimeric xylosyl-xylose and/or longer oligomeric xylose side chains attached to a galacturonan and closely associated to hairy rhamno-galacturonan domains are essential patterns that could determine the biological activity of pectins.

Bioactive capsular polysaccharide from the thermophilic cyanophyte/cyanobacterium Mastigocladus laminosus - cytotoxic properties.

The capsular polysaccharide produced by the thermophilic blue green alga/cyanobacterium Mastigocladus laminosus was tested for its cytotoxic activity against the A431 human epidermoid carcinoma cell line. This polysaccharide inhibited the proliferation of A431 cells in a dose-dependent manner with an IC (50) value of 50 microg mL (-1). In addition, this polysaccharide strongly inhibited A431 cell migration and invasion. Preliminary experiments showing that secretion of metalloproteinases MMP2 and MMP9 by A431 tumour cells was inhibited by this polysaccharide suggest that this mechanism of action could play a role in its anti-migration and anti-invasive properties. Acid hydrolysis of the polysaccharide produced specific oligosaccharides which conserved - at similar concentrations - their cytotoxic, anti-migration and anti-invasion properties; in this case, the mechanism of action was nevertheless uncorrelated to the decrease of metalloproteinase expression.

Structural characterization and cytotoxic properties of a 4-O-methylglucuronoxylan from Castanea sativa.

A glucuronoxylan was purified from a delignified holocellulose alkaline extract of Castanea sativa (Spanish chestnut) and its structure analyzed by means of FT-IR, GC of the per-trimethylsilylated methylglycoside derivatives, and 1H and 13C NMR spectroscopy. The results supported a structure based on a linear polymer of xylopyranose units linked with beta(1-->4) bonds in which, on average, one out of every six units is substituted at C-2 by a 4-O-methylglucuronic acid unit; this structure is typical of a hardwood acidic 4-O-methylglucuronoxylan (MGX) with an estimated degree of polymerization of 200. The MGX from C. sativa inhibited the proliferation of A431 human epidermoid carcinoma cells with an IC50 value of 50 microM. In addition, this xylan inhibited A431 cell migration and invasion. Preliminary experiments showing that secretion of metalloproteinases MMP2 and MMP9 by A431 tumor cells was inhibited by the purified C. sativa MGX strongly suggest that this mechanism of action may play a role in its antimigration and anti-invasive properties.