Structure-antioxidant activity relationship of xylooligosaccharides obtained from carboxyl-reduced glucuronoarabinoxylans from bamboo shoots.

Xylooligosaccharides (XOs) have shown high potential as prebiotics with nutritional and health benefits. In this work, XOs were obtained from highly purified, carboxy-reduced glucuronoarabinoxylans by treatment with Driselase®. The mixtures were fractionated, and the structures were elucidated by methylation analysis and NMR spectroscopy. Antioxidant activity was determined by the methods of DPPH and ß-carotene/linoleic acid. It was found that the most active oligosaccharides (P3 and G3) comprised 4 or 5 xylose units, plus two arabinoses and one 4-O-methylglucose as side chains, their sequence of units was determined. The optimal concentration for their use as antioxidants was 2 mg/mL. The synthetic antioxidant butylated hydroxytoluene (BHT, 0.2 mg/mL) showed a percentage of inhibition 15% higher than P3. Although its concentration was ∼10 times higher, P3 is non-toxic, and could have great advantages as food additive. These results show that pure XOs exert significant antioxidant activity, only due to their carbohydrate nature.

Antioxidant Edible Films Based on Pear Juice and Pregelatinized Cassava Starch: Effect of the Carbohydrate Profile at Different Degrees of Pear Ripeness.

Edible films based on fruit and vegetable purees combined with different food-grade biopolymeric binding agents (e.g., pectin, gelatin, starch, sodium alginate) are recognized as interesting packaging materials that benefit from the physical, mechanical, and barrier properties of biopolymers as well as the sensory and nutritional properties of purees. In the current contribution, edible antioxidant films based on pear juice and pregelatinized cassava starch were developed. In particular, the suitability of using pregelatinized cassava starch for the non-thermal production of these novel edible films was evaluated. In addition, the effects on the films' properties derived from the use of pear juice instead of the complete puree, from the content of juice used, and from the carbohydrate composition associated with the ripening of pears were all studied. The produced films were characterized in terms of their total polyphenol content, water sensitivity, and water barrier, optical, mechanical and antioxidant properties. Results showed that the use of pear juice leads to films with enhanced transparency compared with puree-based films, and that juice concentration and carbohydrate composition associated with the degree of fruit ripeness strongly govern the films' properties. Furthermore, the addition of pregelatinized cassava starch at room temperature discloses a significant and favorable impact on the cohesiveness, lightness, water resistance, and adhesiveness of the pear-juice-based films, which is mainly attributed to the effective interactions established between the starch macromolecules and the juice components.

Diversity of Sulfated Polysaccharides From Cell Walls of Coenocytic Green Algae and Their Structural Relationships in View of Green Algal Evolution.

Seaweeds biosynthesize sulfated polysaccharides as key components of their cell walls. These polysaccharides are potentially interesting as biologically active compounds. Green macroalgae of the class Ulvophyceae comprise sulfated polysaccharides with great structural differences regarding the monosaccharide constituents, linearity of their backbones, and presence of other acidic substituents in their structure, including uronic acid residues and pyruvic acid. These structures have been thoroughly studied in the Ulvales and Ulotrichales, but only more recently have they been investigated with some detail in ulvophytes with giant multinucleate (coenocytic) cells, including the siphonous Bryopsidales and Dasycladales, and the siphonocladous Cladophorales. An early classification of these structurally heterogeneous polysaccharides was based on the presence of uronic acid residues in these molecules. In agreement with this classification based on chemical structures, sulfated polysaccharides of the orders Bryopsidales and Cladophorales fall in the same group, in which this acidic component is absent, or only present in very low quantities. The cell walls of Dasycladales have been less studied, and it remains unclear if they comprise sulfated polysaccharides of both types. Although in the Bryopsidales and Cladophorales the most important sulfated polysaccharides are arabinans and galactans (or arabinogalactans), their major structures are very different. The Bryopsidales produce sulfated pyruvylated 3-linked ß-d-galactans, in most cases, with ramifications on C6. For some species, linear sulfated pyranosic ß-l-arabinans have been described. In the Cladophorales, also sulfated pyranosic ß-l-arabinans have been found, but 4-linked and highly substituted with side chains. These differences are consistent with recent molecular phylogenetic analyses, which indicate that the Bryopsidales and Cladophorales are distantly related. In addition, some of the Bryopsidales also biosynthesize other sulfated polysaccharides, i.e., sulfated mannans and sulfated rhamnans. The presence of sulfate groups as a distinctive characteristic of these biopolymers has been related to their adaptation to the marine environment. However, it has been shown that some freshwater algae from the Cladophorales also produce sulfated polysaccharides. In this review, structures of sulfated polysaccharides from bryopsidalean, dasycladalean, and cladophoralean green algae studied until now are described and analyzed based on current phylogenetic understanding, with the aim of unveiling the important knowledge gaps that still exist.

Glucuronoarabinoxylans and other cell wall polysaccharides from shoots of Guadua chacoensis obtained by extraction in different conditions.

Guadua is a genus of woody bamboos, native to the American continent, which comprises economically important species. Cell wall polysaccharides from young shoots of Guadua chacoensis showed glucuronoarabinoxylans (GAX) as the major hemicellulosic components (65%, of the recovered carbohydrates by extraction with aqueous solutions), which were obtained in major quantities with 1 M KOH (molar ratio Xyl:Ara:GlcA, 100:28:8). AGP and pectin polymers, as well as mixed linkage glucans and xyloglucans were present in smaller amounts (16% and 15%, respectively). Alternative extraction of GAX with solutions of DMSO allowed the characterization of alkali labile substituents, in particular, acetyl groups, which were linked mainly to C3 or, to a lesser extent, to C2 of some of the xylose residues. Besides, small amounts of phenolic compounds, released by saponification, were detected, linked to C5 of some α-l-arabinofuranose units. These results could be relevant for their use as a new resource for the food industry.

Glucuronoarabinoxylans as major cell walls polymers from young shoots of the woody bamboo Phyllostachys aurea.

Young shoots of Phyllostachys aurea showed glucuronoarabinoxylans (GAX) as the major hemicellulosic components, being extracted in major amounts with 1M KOH (ratio Xyl:Ara:GlcA, 100:67:8), but also with water, showing a broad structural variability. Mixed linkage glucans were also present, but in minor amounts, mostly concentrated in the 4M KOH extracts, while pectin polymers were very scarce. Arabinogalactan proteins were an important part of water extracts, determined by the presence of the typical arabinogalactan structures (3- and 6-linked Gal p; terminal and 5-linked Ara f), in addition to small amounts of hydroxyproline (2-3% of total protein) and positive reaction to Yariv's reagent. Morphological and anatomical characteristics of young shoots are described, as well as localization of some cell wall components, and related with chemical analysis. A method for determination of uronic acids as their N-propylaldonamide acetates and separation and quantification by GC/MS was adapted for its use with grass cell wall fractions.

Determination of Substitution Patterns of Galactans from Green Seaweeds of the Bryopsidales.

Sulfated and pyruvylated galactans are the major soluble polysaccharides produced by seaweeds of the Bryopsidales. Their backbones have a complex and variable pattern of substitution which, until now, has only been elucidated for a few species. Methods for determination of sulfate and pyruvic acid content, and chemical strategies to determine their position in the galactan chain are outlined here. These methods can also be applied to other sulfated and/or pyruvylated polysaccharides.

Polysaccharides from the green seaweed Codium decorticatum. Structure and cell wall distribution.

The cell wall polysaccharides from Codium decorticatum and their assembly were studied and these results were compared with those obtained previously for this genus. The water soluble polysaccharides are: (i) Pyruvylated and sulfated 3- and 6-linked ß-D-galactans with sulfate mainly on C-4 and also on C-6. Pyruvate ketals are linked to O-3 and O-4 of terminal ß-D-galactose or O-4 and O-6 of 3-linked ß-D-galactose. (ii) Sulfated 3-linked ß-L-arabinans substituted on C-2 or C-2 and C-4 predominantly with sulfate, but also with single stubs of arabinose, and (iii) 4-linked ß-D-mannans with a low degree of sulfation on C-2. The whole polysaccharide system comprises 6.9% of sulfated polysaccharides and 32.9% of fibrillar polysaccharides, mostly insoluble mannans. By in situ localization it was possible to detect two similar fibrillar layers separated by a zone rich in charged polymers. Besides, arabinogalactan proteins co-localized with the fibrillar components.

Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi.

Arabidopsis heterotrimeric G-protein regulates cell wall defense and resistance to necrotrophic fungi.

The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi. The agb1 mutant impaired in the Gß subunit displays enhanced susceptibility to these pathogens. Gß/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2). Accordingly, we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina. To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance, we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P. cucumerina. This analysis, together with metabolomic studies, demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi, such as the salicylic acid, jasmonic acid, ethylene, abscisic acid, and tryptophan-derived metabolites signaling, as these pathways were not impaired in agb1 and agg1 agg2 mutants. Notably, many mis-regulated genes in agb1 plants were related with cell wall functions, which was also the case in agg1 agg2 mutant. Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants, and that mutant walls had similar FTIR spectratypes, which differed from that of wild-type plants. The data presented here suggest a canonical functionality of the Gß and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition.

Snakin-1 (SN1) is an antimicrobial cysteine-rich peptide isolated from potato (Solanum tuberosum) that was classified as a member of the Snakin/Gibberellic Acid Stimulated in Arabidopsis protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1, potato lines did not show remarkable morphological differences from the wild type; SN1 silencing resulted in reduced height, which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of the adaxial epidermis of mature leaves revealed that silenced lines had 70% to 90% increases in mean cell size with respect to wild-type leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-green fluorescent protein fusion protein was localized in plasma membrane, and bimolecular fluorescence complementation assays revealed that SN1 self-interacted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry, Fourier transform infrared spectroscopy, and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in seven cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism, and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense.

Sulfated ß-d-mannan from green seaweed Codium vermilara.

ß-(1→4)-d-Mannans constitute the major component of the cell wall of seaweeds of the genus Codium and replace cellulose as the major fibrillar component. They were found as major constituents of the hot water extracts of green seaweed Codium vermilara. By anion exchange chromatography of the first hot water extract, a pure sulfated mannan with a molar ratio carbohydrates:sulfate of 2.7:1 was isolated. The sulfate groups are linked to C-2 of 23% of the mannose units, while most of these units are not substituted. This degree of sulfation would explain the higher solubility of the polymer, compared to that of the non-sulfated fibrillar mannan. Taking into account that the fibrillar polysaccharides form two external layers in the cell wall, while the sulfated polymers are forming an amorphous central layer, it is postulated that these sulfated mannans could act as an interphase region between the neutral and acidic layers.

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.