Structural characterization of tissue-specific galactan from flax fibers by 1H NMR and MALDI TOF mass spectrometry

A high-molecular-mass polysaccharide galactan (M 2000 kDa) was isolated from flax at the stage of cell wall thickening of the bast fiber development. The polymer structure was studied by ¹H NMR spectroscopy and MALDI TOF mass spectrometry. It is built up of Gal (59%), Rha (15%), GalA (23%), and Ara (3%) residues. The galactan backbone consists of successively alternating monomer disaccharide units (→ 4GalA1 → 2Rha1 →)_n and is similar in its structure to the backbone of rhamnogalacturonan-1 (RG-I). Rhamnose residues bear in position 4 β-(1 → 4)-galactose side chains of various lengths with a polymerization degree of up to 28 or higher. A part of the side chains have branchings.     

Structural characterization of tissue-specific galactan from flax fibers by 1H NMR and MALDI TOF mass spectrometry

A high-molecular-mass polysaccharide galactan (M 2000 kDa) was isolated from flax at the stage of cell wall thickening of the bast fiber development. The polymer structure was studied by 1H NMR spectroscopy and MALDI TOF mass spectrometry. It is built up of Gal (59%), Rha (15%), GalA (23%), and Ara (3%) residues. The galactan backbone consists of successively alternating monomer disaccharide units (--> 4GalA1 --> 2Rha1 -->)n and is similar in its structure to the backbone of rhamnogalacturonan-1 (RG-I). Rhamnose residues bear in position 4 beta-(1 --> 4)-galactose side chains of various lengths with a polymerization degree of up to 28 or higher. A part of the side chains have branchings.     

The effect of soil drought on the phloem fiber development in long-fiber flax

The effects of soil drought on various stages of phloem fiber development during the period of flax (Linum usitatissimum L.) rapid growth were assessed. The formation of the secondary cell wall was shown to be most retarded. The content of a tissue-specific galactan was reduced especially sharply, and its side chains were changed. Under conditions of pronounced stress-induced plant growth retardation, fiber intrusive growth was suppressed relatively softly: their number on the stem transverse sections was reduced only by 16%. However, this determined irreversible diversity in the fiber length in various stem regions. Such insignificant suppression of intrusive growth under osmotic stress (simultaneously with substantial retardation of plant growth and metabolism inhibition) indicates the functioning of special mechanisms of its regulation.     

Secondary cell-wall assembly in flax phloem fibres: role of galactans

Non-lignified fibre cells (named gelatinous fibres) are present in tension wood and the stems of fibre crops (such as flax and hemp). These cells develop a very thick S2 layer within the secondary cell wall, which is characterised by (1) cellulose microfibrils largely parallel to the longitudinal axis of the cell, and (2) a high proportion of galactose-containing polymers among the non-cellulosic polysaccharides. In this review, we focus on the role of these polymers in the assembly of gelatinous fibres of flax. At the different stages of fibre development, we analyse in detail data based on sugar composition, linkages of pectic polymers, and immunolocalisation of the β-(1→4)-galactans. These data indicate that high molecular-mass gelatinous galactans accumulate in specialised Golgi-derived vesicles during fibre cell-wall thickening. They consist of RG-I-like polymers with side chains of β-(1→4)-linked galactose. Most of them are short, but there are also long chains containing up to 28 galactosyl residues. At fibre maturity, two types of cross-linked galactans are identified, a C–L structure that resembles the part of soluble galactan with long side chains and a C–S structure with short chains. Different possibilities for soluble galactan to give rise to C–L and C–S are analysed. In addition, we discuss the prospect for the soluble galactan in preventing the newly formed cellulose chains from completing immediate crystallisation. This leads to a hypothesis that firstly the secretion of soluble galactans plays a role in the axial orientation of cellulose microfibrils, and secondly the remodelling and cross-linking of pectic galactans are linked to the dehydration and the assembly of S2 layer.     

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.     

Galactosidase of plant fibers with gelatinous cell wall: Identification and localization

Using a comprehensive approach, we have identified a tissue-specific β-galactosidase from flax (Linum usitatissimum L.) phloem fibers forming a gelatinous cell wall. It was found that when fibers started to develop gelatinous cell wall, β-galactosidase gene expression was enhanced.. Using the antibodies against β-galactosidase, we showed that the enzyme was located in flax phloem fibers where it was detected together with tissue-specific galactan in secreted Golgi vesicles and in gelatinous secondary cell wall. Similar β-galactosidase present in gelatinous cell wall of fibers was found in plants belonging to various taxa and produced by different meristems; these data presume the identical mechanisms of gelatinous cell wall formation and an important role of β-galactosidase. The role of this enzyme in developing the supramolecular structure of gelatinous cell wall is discussed.     

Homofusion of Golgi secretory vesicles in flax phloem fibers during formation of the gelatinous secondary cell wall

The gelatinous type of secondary cell wall is present in tension wood and in phloem fibers of many plants. It is characterized by the absence of xylan and lignin, a high cellulose content and axially orientated microfibrils in the huge S2 layer. In flax phloem fiber, the major non-cellulosic component of such cell walls is tissue-specific galactan, which is tightly bound to cellulose. Ultrastructural analysis of flax fiber revealed that initiation of gelatinous secondary cell wall formation was accompanied by the accumulation of specific Golgi vesicles, which had a characteristic bicolor (dark-light) appearance and were easily distinguishable from vesicles made in different tissues and during the other stages of fiber development. Many of the bicolor vesicles appeared to fuse with each other, forming large vacuoles. The largest observed was 4 mum in diameter. Bicolor vesicles and vacuoles fused with the plasma membrane and spread their content in a characteristic "syringe-like" manner, covering a significant area of periplasm and forming "dark" stripes on the inner wall surface. Both Golgi derivatives and cell wall layers were labeled by LM5 antibody, indicating the presence of tissue- and stage-specific (1-->4)-beta-galactan. We suggest that this specific type of galactan secretion, which allows coverage of a large area of periplasm, is designed to increase the chance of the galactan meeting the cellulose microfibrils while they are still in the process of construction. The membrane fusion machinery of flax fiber must possess special components, which may be crucial for the formation of the gelatinous type cell wall.     

Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side‐chains of rhamnogalacturonan‐I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP‐Gal to growing β‐1,4‐galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP‐α‐d ‐Gal and the transfer of an arabinopyranose from UDP‐β‐l ‐Ara_p to galactan chains. The two substrates share a similar structure, but UDP‐α‐d ‐Gal is the preferred substrate, with a 10‐fold higher affinity. Transfer of Ara_p to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.     

New biocomposites based on bioplastic flax fibers and biodegradable polymers

A new generation of entirely biodegradable and bioactive composites with polylactic acid (PLA) or poly‐ε‐caprolactone (PCL) as the matrix and bioplastic flax fibers as reinforcement were analyzed. Bioplastic fibers contain polyhydroxybutyrate and were obtained from transgenic flax. Biochemical analysis of fibers revealed presence of several antioxidative compounds of hydrophilic (phenolics) and hydrophobic [cannabidiol (CBD), lutein] nature, indicating their high antioxidant potential. The presence of CBD and lutein in flax fibers is reported for the first time. FTIR analysis showed intermolecular hydrogen bonds between the constituents in composite PLA+flax fibers which were not detected in PCL‐based composite. Mechanical analysis of prepared composites revealed improved stiffness and a decrease in tensile strength. The viability of human dermal fibroblasts on the surface of composites made of PLA and transgenic flax fibers was the same as for cells cultured without composites and only slightly lower (to 9%) for PCL‐based composites. The amount of platelets and Escherichia coli cells aggregated on the surface of the PLA based composites was significantly lower than for pure polymer. Thus, composites made of PLA and transgenic flax fibers seem to have bacteriostatic, platelet anti‐aggregated, and non‐cytotoxic effect. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Conformation and mobility of the arabinan and galactan side-chains of pectin

The function of the arabinan and galactan side-chains of pectin remains unknown. We describe 13C NMR experiments designed to yield spectra from the most mobile polymer components of hydrated cell walls isolated from a range of plant species. In pectin-rich cell walls, these corresponded to the pectic side-chains. The arabinan side-chains were in general more mobile than the galactans, but the long galactan side-chains of potato pectin showed high mobility. Due to motional line-narrowing effects these arabinan and galactan chains gave 13C NMR spectra of higher resolution than has previously been observed from 'solid' biopolymers. These spectra were similar to those reported for the arabinan and galactan polymers in the solution state, implying time-averaged conformations resembling those found in solution. The mobility of the highly esterified galacturonan in citrus cell walls overlapped with the lower end of the mobility range characteristic of the pectic side-chains. The cellulose-rich cell walls of flax phloem fibres gave spectra of low intensity corresponding to mobile type II arabinogalactans. Cell walls from oat coleoptiles appeared to contain no polymers as mobile as the pectic arabinans and galactans in primary cell walls of the other species examined. These properties of the pectic side-chains suggest a role in interacting with water.     

Residue specific hydration of primary cell wall potato pectin identified by solid-state 13C single-pulse MAS and CP/MAS NMR spectroscopy

Hydration of rhamnogalacturonan-I (RG-I) derived from potato cell wall was analyzed by (13)C single-pulse (SP) magic-angle-spinning (MAS) and (13)C cross-polarization (CP) MAS nuclear magnetic resonance (NMR) and supported by (2)H SP/MAS NMR experiments. The study shows that the arabinan side chains hydrate more readily than the galactan side chains and suggests that the overall hydration properties can be controlled by modifying the ratio of these side chains. Enzymatic modification of native (NA) RG-I provided samples with reduced content of arabinan (sample DA), galactan (sample DG), or both side chains (sample DB). Results of these samples suggested that hydration properties were determined by the length and character of the side chains. NA and DA exhibited similar hydration characteristics, whereas DG and DB were difficult to hydrate because of the less hydrophilic properties of the rhamnose-galacturonic acid (Rha-GalA) backbone in RG-I. Potential food ingredient uses of RG-I by tailoring of its structure are discussed.     

Flavonoid-Peroxidase Reaction as a Detoxification Mechanism of Plant Cells against H2O2

To develop antibody probes for the neutral side chains of pectins, antisera were generated to a pectic galactan isolated from tomato (Lycopersicon esculentum) pericarp cell walls and to a (1[->]4)-[beta]-galactotetraose-bovine serum albumin neoglycoprotein. The use of these two antisera in immunochemical assays and immunolocalization studies indicated that they had very similar specificities. A monoclonal antibody (LM5) was isolated and characterized subsequent to immunization with the neoglycoprotein. Hapten inhibition studies revealed that the antibody specifically recognized more than three contiguous units of (1[->]4)-[beta]-galactosyl residues. The antigalactan antibody was used to immunolocalize the galactan side chains of pectin in tomato fruit pericarp and tomato petiole cell walls. Although the LM5 epitope occurs in most cell walls of the tomato fruit, it was absent from both the locular gel and the epidermal and subepidermal cells. Furthermore, in contrast to other anti-pectin antibodies, LM5 did not label the cell wall thickenings of tomato petiole collenchyma.     

The Inhibitory Effects of a Rhamnogalacturonan Ι (RG-I) Domain from Ginseng Pectin on Galectin-3 and Its Structure-Activity Relationship

Pectin has been shown to inhibit the actions of galectin-3, a β-galactoside-binding protein associated with cancer progression. The structural features of pectin involved in this activity remain unclear. We investigated the effects of different ginseng pectins on galectin-3 action. The rhamnogalacturonan I-rich pectin fragment, RG-I-4, potently inhibited galectin-3-mediated hemagglutination, cancer cell adhesion and homotypic aggregation, and binding of galectin-3 to T-cells. RG-I-4 specifically bound to the carbohydrate recognition domain of galectin-3 with a dissociation constant of 22.2 nm, which was determined by surface plasmon resonance analysis. The structure-activity relationship of RG-I-4 was investigated by modifying the structure through various enzymatic and chemical methods followed by activity tests. The results showed that (a) galactan side chains were essential to the activity of RG-I-4, whereas arabinan side chains positively or negatively regulated the activity depending on their location within the RG-I-4 molecule. (b) The activity of galactan chain was proportional to its length up to 4 Gal residues and largely unchanged thereafter. (c) The majority of galactan side chains in RG-I-4 were short with low activities. (d) The high activity of RG-I-4 resulted from the cooperative action of these side chains. (e) The backbone of the molecule was very important to RG-I-4 activity, possibly by maintaining a structural conformation of the whole molecule. (f) The isolated backbone could bind galectin-3, which was insensitive to lactose treatment. The novel discovery that the side chains and backbone play distinct roles in regulating RG-I-4 activity is valuable for producing highly active pectin-based galectin-3 inhibitors.     

Elucidating the effects of laccase-modifying compounds treatments on bast and core fibers in flax pulp

Laccases in combination with various chemical compounds have been tested with a view to obtain environmental friendly, high‐value paper products from unbleached flax pulp, which is currently being assessed as a raw material for biotechnological innovation. With the aim of better understanding the effects of violuric acid (VA) and p‐coumaric acid (PCA) on flax pulp, changes in the chemical composition of the two major fiber types it contains were assessed. Following classification, the initial pulp was split into two fractions according to fiber size, namely: bast (long) fibers and core (short) fibers. Fiber size was found to significantly influence the properties of pulp and it response to various laccase treatments. The laccase‐PCA treatment substantially increased kappa number (KN) and color in both fiber fractions, which suggests grafting of the phenolic compound onto fibers. On the other hand, the laccase‐VA treatment produced long fibers with a low lignin content (KN = 1.3) and a high brightness (5% points higher than for the control fraction), which testifies to its bleaching efficiency. Both biotreatments produced long fibers containing highly crystalline cellulose and caused HexA removal from global and short fibers. On the other hand, the laccase treatments caused no morphological changes in the fibers, the integrity of which was largely preserved. As shown here, laccase acts as polymerization agent with PCA and as delignification agent with VA; also, the two enzymes systems act differently on bast and core fibers. Biotechnol. Bioeng. 2012;109: 225–233. © 2011 Wiley Periodicals, Inc.     

Characterization of a biologically active arabinogalactan from the leaves of Plantago major L.

PMIa is a Type II arabinogalactan with anti-complementary activity isolated from the leaves of Plantago major L. It has a molecular weight of 77000–80000 Da and consists of arabinose (38%), galactose (49%), rhamnose (6%), galacturonic acid (7%) and 1.5% protein with hydroxyproline, alanine and serine as the main amino acids. Characterization of PMIa by methylation and GC-MS, methanolysis and GC, Smith degradation, weak acid hydrolysis, ¹³C-NMR, ¹H-NMR, two-dimensional heteronuclear NMR and DEPT show that it consists of 1,3-linked galactan chains with 1,6-linked galactan side chains attached to position 6. The side chains are further branched in position 3 with 1,3-linked galactose residues which have 1,6-linked galactose attached to position 6; these 1,3- and 1,6-linked galactose chains altogether probably form a network. Terminal and 1,5-linked arabinose in furanose form are attached to the galactan mainly through position 3 of the 1,6-linked galactose side chains.     

Relatedness of Indian Flax Genotypes (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.): An Inter-Simple Sequence Repeat (ISSR) Primer Assay

The objective of this study was to analyze the genetic relationships, using PCR-based ISSR markers, among 70 Indian flax (Linum usitatissimum L.) genotypes actively utilized in flax breeding programs. Twelve ISSR primers were used for the analysis yielding 136 loci, of which 87 were polymorphic. The average number of amplified loci and the average number of polymorphic loci per primer were 11.3 and 7.25, respectively, while the percent loci polymorphism ranged from 11.1 to 81.8 with an average of 63.9 across all the genotypes. The range of polymorphism information content scores was 0.03–0.49, with an average of 0.18. A dendrogram was generated based on the similarity matrix by the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), wherein the flax genotypes were grouped in five clusters. The Jaccard’s similarity coefficient among the genotypes ranged from 0.60 to 0.97. When the omega-3 alpha linolenic acid (ALA) contents of the individual genotypes were correlated with the clusters in the dendrogram, the high ALA containing genotypes were grouped in two clusters. This study identified SLS 50, Ayogi, and Sheetal to be the most diverse genotypes and suggested their use in breeding programs and for developing mapping populations.     

Purification and characterization of an endo-beta-(1-->6)-galactanase from Trichoderma viride

An endo-beta-(1-->6)-galactanase from Onozuka R-10, a commercial cellulase preparation from Trichoderma viride, was purified 57-fold. Apparent Mr values of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were 47,000 and 17,000, respectively. The enzyme was assayed with a galactan from Prototheca zopfii, which has a high proportion of beta-(1-->6)-linked galactosyl residues. It exhibited maximal activity toward the galactan at pH 4.3. The enzyme hydrolyzed specifically beta-(1-->6)-galactooligosaccharides with a degree of polymerization higher than 3 and their acidic derivatives with 4-O-methyl-glucosyluronic or glucosyluronic groups at the nonreducing terminals. The methyl beta-glycoside of beta-(1-->6)-galactohexaose was degraded to reducing galactooligomers with a degree of polymerization 2-5 as the products at the initial stage of hydrolysis, and galactose and galactobiose at the final stage, indicating that the enzyme can be classified as an endo-galactanase. The extent of hydrolysis of the carbohydrate portion of a radish root arabinogalactan-protein (AGP) increased when alpha-L-arabinofuranosyl residues attached to beta-(1-->6)-linked galactosyl side chains of the AGP were removed in advance. The enzyme released galactose, beta-(1-->6)-galactobiose, and 4-O-methyl-beta-glucuronosyl-(1-->6)-galactose as major hydrolysis products when allowed to act exhaustively on the modified AGP.     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

Modification of polysaccharides from callus culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (M.) G. using carbohydrases <Emphasis Type="Italic">in vitro</Emphasis>

Polysaccharides (pectin and intracellular and extracellular arabinogalactans) were isolated from campion callus culture cultivated on medium with varied concentrations of pectinase and beta-galactosidase. A decrease in contents of arabinose residues in pectin and arabinogalactans and of galactose residues in arabinogalactans was associated with an increase in the activities of alpha-L-arabinofuranosidase and beta-galactosidase upon addition of pectinase into the medium. Pectinase destroyed the high-molecular-weight (more than 300 kD) fraction of pectin and decreased the content of galacturonic acid residues. alpha-L-Arabinofuranosidase transformed arabinogalactan into galactan, and galactan was destroyed under the influence of galactosidase. The contents of arabinogalactan and/or galactan in the cells were decreased, and it was released into the culture medium. Pectin samples with low contents of arabinose and galactose in the side chains and galactan samples were obtained from the callus grown on the medium with beta-galactosidase. Cultivation of the plant cells on medium containing carbohydrases resulted in modification of pectin and arabinogalactan of the cell walls.     

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives

 The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. × P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours. Received: 12 June 1999 / Accepted: 20 October 1999