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Hempseed (Cannabis sativa L.) is a valuable source of oil and a quality vegetable protein source, but little is known about its fiber composition. This study provides the most extensive characterization of hempseed cell wall polysaccharides to date. Quantification of total dietary fiber (TDF) and acetyl bromide soluble lignin (ABSL), monosaccharide composition through two complimentary hydrolysis methods (Saeman hydrolysis and methanolysis), and linkage analysis via derivatization of monosaccharides to partially methylated alditol acetates (PMAAs) were done. More detailed profiling of hempseed xyloglucans and pectic arabinans and galactans was performed via targeted enzymatic generation and quantification of characteristic oligosaccharides. Hempseed's cell wall polysaccharide profile is consistent with the primary cell walls of dicots, but it is particularly abundant in linear xylans, presumably from secondary cell walls. The hempseed water-insoluble cell wall material was mostly composed of cellulose and xylans, but pectins (including arabinans with a low degree of branching and galactans), xyloglucans with L- and F-motifs and a relatively low substitution rate, and mannans were also present. These data show that hempseed is a good source of complex dietary fiber for human diets and a potential fiber additive for herbivorous livestock diets. Potential consequences of these carbohydrate structural details on hempseed's microbial fermentability are discussed. [ABSTRACT FROM AUTHOR]

During the development of legume-rhizobial symbiosis, the process of rhizobia internalization depends on the modification of the plant-microbial surfaces and, first of all, on the remodeling of cell walls of both plant cells and infection threads. The components of cell walls, intercellular matrix and plasma and symbiosome membranes in nitrogen-fixing nodules of two legume species belonging to different types—determinate (Glycine max L. Merrill) and indeterminate (Galega orientalis Lam.)—were studied. As a result, the localization and distribution of different types of pectins, fucosylated xyloglucans, cell wall proteins and arabinogalactan proteins were revealed. Species-specific features of the symbiotic interface were identified, namely the presence of the linear galactan side chain of rhamnogalacturonan I in the infection thread walls, the rhamnogalacturonan I backbone, Ca2+-linked homogalacturonan, and an increase in the labeling of low methylesterified homogalacturonan in the cell walls of uninfected cells in the indeterminate nodules of G. orientalis; the presence of the galactan side chain of rhamnogalacturonan I in the cell walls of uninfected cells and the predominant accumulation of fucosylated xyloglucan in the infection thread walls in the determinate nodules of G. max. In nodules of both species, arabinans (cross-linked with rhamnogalacturonan I and as part of arabinogalactan proteins) play a major role in the development of infection structures, in particular symbiosomes, namely, they are detected at early stages of symbiosome differentiation. [ABSTRACT FROM AUTHOR]

Many insect-induced galls are considered complex structures due to their tissue compartmentalization and multiple roles performed by them. The current study investigates the complex interaction between Nothofagus obliqua host plant and the hymenopteran gall-inducer Espinosa nothofagi, focusing on cell wall properties and cytological features. The E. nothofagi galls present an inner cortex with nutritive and storage tissues, as well as outer cortex with epidermis, chlorenchyma, and water-storing parenchyma. The water-storing parenchyma cells are rich in pectins, heteromannans, and xyloglucans in their walls, and have large vacuoles. Homogalacturonans contribute to water retention, and periplasmic spaces function as additional water reservoirs. Nutritive storage cell walls support nutrient storage, with plasmodesmata facilitating nutrient mobilization crucial for larval nutrition. Their primary and sometimes thick secondary cell walls support structural integrity and act as a carbon reserve. The absent labeling of non-cellulosic epitopes indicates a predominantly cellulosic nature in nutritive cell walls, facilitating larval access to lipid, protein, and reducing sugar-rich contents. The nutritive tissue, with functional chloroplasts and high metabolism-related organelles, displays signs of self-sufficiency, emphasizing its role in larval nutrition and cellular maintenance. Overall, the intricate cell wall composition in E. nothofagi galls showcases adaptations for water storage, nutrient mobilization, and larval nutrition, contributing significantly to our understanding of plant–insect interactions. [ABSTRACT FROM AUTHOR]

In response to the restrictions imposed by their epiphytic habit, orchids have developed structural traits that allow greater efficiency in water uptake and use, such as a complex adventitious root system with velamen. The composition of cell wall of this specialized epidermis can be altered according to the substrate to which it is fixed, influencing wall permeability, absorption, and storage of water in roots. The current study aimed to evaluate the cell wall composition of adventitious roots of Vanilla phaeantha (Orchidaceae) that grow attached to the phorophyte, fixed in the soil, or hung free. Immunocytochemical analyses were used to determine the protein, hemicellulose, and pectin composition of the cell walls of aerial and terrestrial roots. We observed that pectins are present in the different tissues of the aerial roots, while in the terrestrial roots, they are concentrated in the cortical parenchyma. The deposition of xyloglucans, extensins, and arabinogalactans was greater in the epidermis of the free side of the roots attached to the phorophyte. The strong labeling of pectins in aerial roots may be related to the influx of water and nutrients, which are generally scarce in this environment. The arrangement of hemicelluloses and proteins with the pectins may be associated with increased cell rigidity and sustainability, a feature of interest for the aerial roots. In summary, the habit of roots can interfere with the non-cellulosic composition of the cell walls of V. phaeantha, possibly related to changes in cell functionality. [ABSTRACT FROM AUTHOR]

α-Xylosidases release the α-d-xylopyranosyl side chain from di- and oligosaccharides derived from xyloglucans and are involved in xyloglucan degradation. In this study, an extracellular α-xylosidase, named AxyB, is identified and characterized in Aspergillus oryzae. AxyB belongs to the glycoside hydrolase family 31 and releases d-xylose from isoprimeverose (α-d-xylopyranosyl-(1 → 6)-d-glucopyranose) and xyloglucan oligosaccharides. In the hydrolysis of xyloglucan oligosaccharides (XLLG, Glc4Xyl3Gal2 nonasaccharide; XLXG/XXLG, Glc4Xyl3Gal1 octasaccharide; and XXXG, Glc4Xyl3 heptasaccharide), AxyB releases one molecule of the xylopyranosyl side chain attached to the non-reducing end of the β-1,4-glucan main chain of these xyloglucan oligosaccharides to yield GLLG (Glc4Xyl2Gal2), GLXG/GXLG (Glc4Xyl2Gal1), and GXXG (Glc4Xyl2). A. oryzae has both extracellular and intracellular α-xylosidase, suggesting that xyloglucan oligosaccharides are degraded by a combination of isoprimeverose-producing oligoxyloglucan hydrolase and intracellular α-xylosidase and a combination of extracellular α-xylosidase and β-glucosidase(s) in A. oryzae. Key points: • An extracellular α-xylosidase, AxyB, is identified in Aspergillus oryzae. • AxyB releases the xylopyranosyl side chain from xyloglucan oligosaccharides. • Different sets of glycosidases degrade xyloglucan oligosaccharides in A. oryzae. [ABSTRACT FROM AUTHOR]

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis. Xyloglucans are polysaccharides found in plant cell walls. Here, the authors describe the xyloglucan depolymerization machinery of phytopathogenic Xanthomonas bacteria, and show that sugars released by this system induce the expression of key virulence factors driving pathogenesis. [ABSTRACT FROM AUTHOR]

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum. Mikkelsen et al. demonstrate molecular and cellular evidence of the evolution of xyloglucan (XyG), a structural component of the cell walls of most land plants, in charophyte algae. This study describes the structure of XyG in charophyte algae, including identification of fucosylated XyG, and furthermore identifies orthologs required to produce XyG. [ABSTRACT FROM AUTHOR]

In the current study, effects of chitosan oligomers (CHI-OM) with different polymerization degrees (DP) between 2 and 15, and polymeric chitosan (CHI-P) with a DP of 70 were compared with kinetin (KIN), 6-benzylaminopurine (BAP), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and jasmonic acid (JAS) on the structure and composition of several biomolecules in the root system of Serapias vomeracea. Evaluation of molecular alterations in the root system of S. vomeracea through an infrared spectroscopic approach provided insight into the differentiation between action mechanisms of chitosan and commonly used plant growth regulators. The results revealed that CHI-P and JAS treatments at low concentrations might enhance the lignin content in the cell walls. Also, JAS, CHI-OM, and CHI-P treatments enhanced cell wall lignification. The water-associated cellulose content of the cell walls was affected by the DP of chitosan. Membrane lipid stabilization and protein content were enhanced after CHI-P, JAS, and KIN treatments, while BAP and CHI-OM treatments triggered lipid, protein, and cell wall polysaccharide synthesis. In particular, CHI-OM treatment also enhanced rhamnogalacturonan and β-galactan content as well as xyloglucans and glucomannans, while the auxin treatments had a similar impact only on glucomannan content. These findings suggest that chitosan may show either purine-based cytokinin-like or lipid-based jasmonic acid-like activity in the root system of S. vomeracea. Additionally, depending on its degree of polymerization, the effects of chitosan may vary on root system development in S. vomeracea. [ABSTRACT FROM AUTHOR]

Key message: CsSh5.1, which controls hypocotyl elongation under high temperature conditions in cucumber, was mapped to a 57.1 kb region on chromosome 5 containing a candidate gene encoding a xyloglucan galactosyltransferase. Hypocotyl growth is a vital process in seedling establishment. Hypocotyl elongation after germination relies more on longitudinal cell elongation than cell division. Cell elongation is largely determined by the extensibility of the cell wall. Here, we identified a spontaneous mutant in cucumber (Cucumis sativus L.), sh5.1, which exhibits a temperature-insensitive short hypocotyl phenotype. Genetic analysis showed that the phenotype of sh5.1 was controlled by a recessive nuclear gene. CsSh5.1 was mapped to a 57.1 kb interval on chromosome 5, containing eight predicted genes. Sequencing analysis revealed that the Csa5G171710 is the candidate gene of CsSh5.1, which was further confirmed via co-segregation analysis and genomic DNA sequencing in natural cucumber variations. The result indicated that hypocotyl elongation might be controlled by this gene. CsSh5.1 encodes a xyloglucan galactosyltransferase that specifically adds galactose to xyloglucan and forms galactosylated xyloglucans, which determine the strength and extensibility of the cell walls. CsSh5.1 expression in wild-type (WT) hypocotyl was significantly higher than that in sh5.1 hypocotyl under high temperature, suggesting its important role in hypocotyl cell elongation under high temperature. The identification of CsSh5.1 is helpful for elucidating the function of xyloglucan galactosyltransferase in cell wall expansion and understanding the mechanism of hypocotyl elongation in cucumber. [ABSTRACT FROM AUTHOR]

Aspergillus species are closely associated with humanity through fermentation, infectious disease, and mycotoxin contamination of food. Members of this genus produce various enzymes to degrade plant polysaccharides, including starch, cellulose, xylan, and xyloglucan. This review focus on the machinery of the xyloglucan degradation using glycoside hydrolases, such as xyloglucanases, isoprimeverose-producing oligoxyloglucan hydrolases, and α-xylosidases, in Aspergillus species. Some xyloglucan degradation-related glycoside hydrolases are well conserved in this genus; however, other enzymes are not. Cooperative actions of these glycoside hydrolases are crucial for xyloglucan degradation in Aspergillus species. Key points: •Xyloglucan degradation-related enzymes of Aspergillus species are reviewed. •Each Aspergillus species possesses a different set of glycoside hydrolases. •The machinery of xyloglucan degradation of A. oryzae is overviewed. [ABSTRACT FROM AUTHOR]

The dynamics of cell wall polysaccharides may modulate the cell wall mechanics and thus control the expansion growth of plant cells. The unique composition of type II primary cell wall characteristic of grasses suggests that they employ specific mechanisms for cell enlargement. We characterized the transcriptomes in five zones along maize root, clustered the expression of genes for numerous glycosyltransferases and performed extensive immunohistochemical analysis to relate the changes in cell wall polysaccharides to critical stages of cell development in Poaceae. Specific patterns of cell wall formation differentiate the initiation, realization and cessation of elongation growth. Cell walls of meristem and early elongation zone represent a mixture of type I and type II specific polysaccharides. Xyloglucans and homogalacturonans are synthesized there actively together with mixed-linkage glucans and glucuronoarabinoxylans. Rhamnogalacturonans-I with the side-chains of branched 1,4-galactan and arabinan persisted in cell walls throughout the development. Thus, the machinery to generate the type I primary cell wall constituents is completely established and operates. The expression of glycosyltransferases responsible for mixed-linkage glucan and glucuronoarabinoxylan synthesis peaks at active or late elongation. These findings widen the number of jigsaw pieces which should be put together to solve the puzzle of grass cell growth. [ABSTRACT FROM AUTHOR]

Rhizobia are nitrogen-fixing soil bacteria that can infect legume plants to establish root nodules symbiosis. To do that, a complex exchange of molecular signals occurs between plants and bacteria. Among them, rhizobial Nops (Nodulation outer proteins), secreted by a type III secretion system (T3SS) determine the host-specificity for efficient symbiosis with plant roots. Little is known about the molecular function of secreted Nops (also called effectors (T3E)) and their role in the symbiosis process. We performed the structure-function characterization of NopAA, a T3E from Sinorhizobium fredii by using a combination of X-ray crystallography, biochemical and biophysical approaches. This work displays for the first time a complete structural and biochemical characterization of a symbiotic T3E. Our results showed that NopAA has a catalytic domain with xyloglucanase activity extended by a N-terminal unfolded secretion domain that allows its secretion. We proposed that these original structural properties combined with the specificity of NopAA toward xyloglucan, a key component of root cell wall which is also secreted by roots in the soil, can give NopAA a strategic position to participate in recognition between bacteria and plant roots and to intervene in nodulation process. [ABSTRACT FROM AUTHOR]

Under the effect of disturbances, like unbalanced stem, but also during normal development, poplar trees can develop a specific secondary xylem, called "tension wood" (TW), which is easily identifiable by the presence of a gelatinous layer in the secondary cell walls (SCW) of the xylem fibers. Since TW formation was mainly performed on 2-year-old poplar models, an in vitro poplar that produces gelatinous fibers (G-fibers) while offering the same experimental advantages as herbaceous plants has been developed. Using specific cell wall staining techniques, wood structural features and lignin/cellulose distribution were both detailed in cross-sections obtained from the curved stem part of in vitro poplars. A supposed delay in the SCW lignification process in the G-fibers, along with the presence of a G-layer, could be observed in the juvenile plants. Moreover, in this G-layer, the immunolabeling of various polymers carried out in the SCW of TW has allowed detecting crystalline cellulose, arabinogalactans proteins, and rhamnogalacturonans I; however, homogalacturonans, xylans, and xyloglucans could not be found. Interestingly, extensins were detected in this typical adaptative or stress-induced structure. These observations were corroborated by a quantitation of the immunorecognized polymer distribution using gold particle labeling. In conclusion, the in vitro poplar model seems highly convenient for TW studies focusing on the implementation of wall polymers that provide the cell wall with greater plasticity in adapting to the environment. [ABSTRACT FROM AUTHOR]

Main conclusion: Galactan turnover occurs during cell elongation and affects the cell wall xyloglucan structure which is involved in the interaction between cellulose and xyloglucan.β-(1,4)-Galactan is one of the main side chains of rhamnogalacturonan I. Although the specific function of this polymer has not been completely established, it has been related to different developmental processes. To study β-(1,4)-galactan function, we have generated transgenic Arabidopsis plants overproducing chickpea βI-Gal β-galactosidase under the 35S CaMV promoter (35S::βI-Gal) to reduce galactan side chains in muro. Likewise, an Arabidopsis double loss-of-function mutant for BGAL1 and BGAL3 Arabidopsis β-galactosidases (bgal1/bgal3) has been obtained to increase galactan levels. The characterization of these plants has confirmed the role of β-(1,4)-galactan in cell growth, and demonstrated that the turnover of this pectic side chain occurs during cell elongation, at least in Arabidopsis etiolated hypocotyls and floral stem internodes. The results indicate that BGAL1 and BGAL3 β-galactosidases act in a coordinate way during cell elongation. In addition, this work indicates that galactan plays a role in the maintenance of the cell wall architecture during this process. Our results point to an involvement of the β-(1,4)-galactan in the xyloglucan structure and the interaction between cellulose and xyloglucan. [ABSTRACT FROM AUTHOR]

The role of xyloglucan endotransglycosylases in the regulation and promotion of plant growth in response to such widespread stress factors as drought, salinization, and hypothermia remains poorly understood. The tXET-B2 (SlXTH10) gene encodes one of the xyloglucan endotransglycosylases of tomato, which is most closely related in the nucleotide sequence to the AtXTH15 and AtXTH16 genes of Arabidopsis thaliana. At present, the specific functions of the tXET-B2 gene, as well as of its homologs, AtXTH15 and AtXTH16, remain obscure. To study the role of tXET-B2 in the regulation of growth and adaptation to abiotic stress factors, transgenic tobacco plants with estradiol-inducible expression of the tomato tXET-B2 gene were generated. Overexpression of this gene promoted tobacco root growth in a medium containing 50 mM NaCl. Under drought conditions, exogenous treatment with estradiol resulted in a considerable increase in fresh and dry weight in many of the studied transgenic lines. Under normal conditions, as well as under salinization and hypothermia stress, such positive effect was detected only for some transgenic lines. The obtained data point to the possibility of using genetically engineered constructs of the tXET-B2 gene to correct growth parameters of transgenic plants under the influence of stress factors. [ABSTRACT FROM AUTHOR]

Main conclusion A large-scale bioinformatics analysis revealed the origin and evolution of GT47 gene family, and identified two clades of intron-poor genes with putative functions in drought stress responses and seed development in maize. Glycosyltransferase family 47 (GT47) genes encode ß-galactosyltransferases and ß-glucuronyltransferases that synthesize pectin, xyloglucans and xylan, which are important components of the plant cell wall. In this study, we performed a systematic and large-scale bioinformatics analysis of GT47 gene family using 352 GT47 proteins from 15 species ranging from cyanobacteria to seed plants. The analysis results showed that GT47 family may originate in cyanobacteria and expand along the evolutionary trajectory to moss. Further analysis of 47 GT47 genes in maize revealed that they can divide into five clades with diverse exon-intron structures. Among these five clades, two were mainly composed with intron-poor genes, which may originate in the moss. Gene duplication analysis revealed that the expansion of GT47 gene family in maize was significantly driven from tandem duplication events and segmental duplication events. Significantly, almost all duplicated genes are intron-poor genes. Expression analysis indicated that several intron-poor GT47 genes may be involved in the drought stress response and seed development in maize. This work provides insight into the origin and evolutionary process, expansion mechanisms and expression patterns of GT47 genes, thus facilitating their functional investigations in the future. [ABSTRACT FROM AUTHOR]

Main conclusion : Unlike most plant cell walls, the five consecutive walls laid down during spermatogenesis in the model fern Ceratopteris contain sparse cellulose, lack pectin and are enriched with callose and hemicelluloses. Seed-free plants like bryophytes and pteridophytes produce swimming male gametes for sexual reproduction. During spermatogenesis, unique walls are formed that are essential to the appropriate development and maturation of the motile gametes. Other than the detection of callose and general wall polysaccharides in scattered groups, little is known about the sequence of wall formation and the composition of these walls during sperm cell differentiation in plants that produce swimming sperm. Using histochemistry and immunogold localizations, we examined the distribution of callose, cellulose, mannan and xylan-containing hemicelluloses, and homogalacturonan (HG) pectins in the special walls deposited during spermatogenesis in Ceratopteris. Five walls are produced in sequence and each has a unique fate. The first wall (W1) contains callose and sparse xylan-containing hemicelluloses. Wall two (W2) is thin and composed of cellulose crosslinked by xylan-containing hemicelluloses. The third wall (W3) is thick and composed entirely of callose, and the fourth wall (W4) is built of cellulose heavily crosslinked by galactoxyloglucan hemicelluloses. Wall five (W5) is an arabinogalactan protein (AGP)-rich matrix in which the gamete changes shape and multiple flagella elongate. We detected no esterified or unesterified HG pectins in any of the walls laid down during spermatogenesis. To consider evolutionary modifications in cell walls associated with motile gametes, comparisons are presented with male gametophyte and spermatogenous cell walls across plant groups. [ABSTRACT FROM AUTHOR]

A xyloglucanase of the GH74 family was identified in the thermophilic fungus strain Myceliophthora thermophila VKPM F-244, and its gene sequence was optimized for cloning and expression in Pichia pastoris. The recombinant xyloglucanase MtXgh74 exhibited the highest activity toward tamarind seed xyloglucan with a K value of 0.51 ± 0.06 mg/mL. The activities on barley β-glucan and carboxymethylcellulose were about 4 and 2%, respectively, compared to xyloglucan. Maximum xyloglucanase activity was observed at 70-75 °C and pH 6.5. After pre-incubation at 50 °C, pH 6.0 for 3 h, the enzyme retained 100% of its activity. The half-life of MtXgh74 at 60 °C, pH 6.0 was 40 min. In P. pastoris, MtXgh74 was produced in glycosylated form. The enzyme production in a 1 L bioreactor resulted in a yield of 118 U/mL or 5.3 g/L after 51 h fermentation. Kinetic studies of the hydrolysis product formation suggest that MtXgh74 has an endo-processive mode of action. The final products were the standard xyloglucan building blocks XXXG, XXLG, XLXG, and XLLG. Additionally, MtXgh74 hydrolyzed various linkages within the xyloglucan building blocks XXXG, XXLG, and XLXG (except XLLG) producing diverse low molecular weight oligosaccharides which may be identified by MALDI-TOF as XG, XX, XXG/GXX/XGX, XXX, LG, LX/XL, XLX/XXL, LLG, GXXXG, GXLLG, XLLGX. The unique combination of different activities within one enzyme along with its high thermostability and specificity toward xyloglucan makes MtXgh74 a promising candidate enzyme for industrial applications. [ABSTRACT FROM AUTHOR]

Capsinoids are non-pungent analogues of capsaicinoids in pepper ( Capsicum spp). The absence of pungency, in addition to their biological activities similar to that of capsaicinoids such as anti-inflammatory, antimicrobial, and antioxidant properties, makes capsinoids an excellent option for increasing use in human and animal nutrition, as well as health and pharmaceutical industries. There are only few sources of pepper producing capsinoids, and one of them (accession 509-45-1), Capsicum annuum L., is a potential source for increasing capsinoids content using strategies as controlled elicitation during plant production in the greenhouse. In this research we evaluated the effect of weekly and one-day-before-harvest foliar applications of hydrogen peroxide, salicylic acid and a xyloglucan oligosaccharide on the concentration of capsiate in fruits of this pepper accession, as well as the gene expression of phenylalanine ammonia-lyase ( pal), putative aminotransferase ( pamt), capsaicin synthase ( at3) and β-keto acyl synthase ( kas). Results showed that the two tested concentrations of HO significantly increased capsiate content and gene expression associated with capsaicinoids ( pamt, at3 and kas) and the phenylpropanoids ( pal) pathways. Plant yield was not affected using this induction strategy. Our results indicated that the pre-harvest and weekly application of hydrogen peroxide and xyloglucan oligosaccharide improved production of capsiate in C. annuum L. [ABSTRACT FROM AUTHOR]

Key message: DkXTH1 promoted cell elongation and more strength to maintain structural integrity by involving in cell wall assembly, thus enhanced tolerance to abiotic stress with broader phenotype in transgenic plants. Abstract: Xyloglucan endotransglucosylase/hydrolase (XTH) is thought to play a key role in cell wall modifications by cleaving and re-joining xyloglucan, and participates in the diverse physiological processes. DkXTH1 was found to peak in immature expanding persimmon fruit, and its higher expression level exhibited along with firmer fruit during storage. In the present study, transgenic Arabidopsis and tomato plants were generated with DkXTH1 constitutively expressed. Overexpression of DkXTH1 enhanced tolerance to salt, ABA and drought stresses in transgenic Arabidopsis plants with respect to root and leaf growth, and survival. Transgenic tomatoes collected at the mature green stage, presented delayed fruit softening coupled with postponed color change, a later and lower ethylene peak, and higher firmness in comparison with the wild-type tomatoes during storage. Furthermore, broader leaves and tomato fruit with larger diameter were gained in transgenic Arabidopsis and tomato, respectively. Most importantly, transgenic plants exhibited more large and irregular cells with higher density of cell wall and intercellular spaces, resulting from the overactivity of XET enzymes involving in cell wall assembly. We suggest that DkXTH1 expression resulted in cells with more strength and thickness to maintain structural integrity, and thus enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants. [ABSTRACT FROM AUTHOR]

Xyloglucan is the most abundant hemicellulose in primary walls of spermatophytes except for grasses. Xyloglucan-degrading enzymes are important in lignocellulosic biomass hydrolysis because they remove xyloglucan, which is abundant in monocot-derived biomass. Fungal genomes encode numerous xyloglucanase genes, belonging to at least six glycoside hydrolase (GH) families. GH74 endo-xyloglucanases cleave xyloglucan backbones with unsubstituted glucose at the −1 subsite or prefer xylosyl-substituted residues in the −1 subsite. In this work, 137 GH74-related genes were detected by examining 293 Eurotiomycete genomes and Ascomycete fungi contained one or no GH74 xyloglucanase gene per genome. Another interesting feature is that the triad of tryptophan residues along the catalytic cleft was found to be widely conserved among Ascomycetes. The GH74 from Aspergillus fumigatus (AfXEG74) was chosen as an example to conduct comprehensive biochemical studies to determine the catalytic mechanism. AfXEG74 has no CBM and cleaves the xyloglucan backbone between the unsubstituted glucose and xylose-substituted glucose at specific positions, along the XX motif when linked to regions deprived of galactosyl branches. It resembles an endo-processive activity, which after initial random hydrolysis releases xyloglucan-oligosaccharides as major reaction products. This work provides insights on phylogenetic diversity and catalytic mechanism of GH74 xyloglucanases from Ascomycete fungi. [ABSTRACT FROM AUTHOR]

This study investigated cellulose production and microstructure variation from six Komagataeibacter strains (ATCC 53524, ATCC 10245, ATCC 23769, ATCC 700178, NBRC 13693 and KTH 5655). Strain KTH 5655 produced the highest cellulose yields (10.39 g/l) after 9 days cultivation. Nuclear magnetic resonance spectroscopy and X-ray diffraction revealed that strain ATCC 23769 synthesised cellulose with the lowest crystallinity and decreased ratio of Iα/Iβ allomorph, whilst strain KTH 5655 produced a relatively ordered cellulose structure. However, the average widths of cellulose ribbons were similar (30-50 nm) for all types of cellulose. Phylogenetic analysis of the 16S rRNA gene indicated that these strains shared a high level of genetic similarity (ranging from 88 to 98%). All strains were used to produce cellulose in the presence of arabinoxylan or xyloglucan as simplified cell wall analogues. Our results provide guidance for the selection of cellulose-producing strains for specific biotechnological and research applications. [ABSTRACT FROM AUTHOR]

Polysaccharides are finding an increasing number of applications in medical and pharmaceutical fields thanks to their biodegradability, biocompatibility, and in some cases bioactivity. Two approaches were applied to use hemicelluloses as crosslinkers to tune the structural and mechanical properties of nanofibrillated cellulose (NFC) hydrogel scaffolds, and thus to investigate the effect of these properties on the cellular behavior during wound healing application. Different types of hemicellulose (galactoglucomannan (GGM), xyloglucan (XG), and xylan) were introduced into the NFC network via pre-sorption (Method I) and in situ adsorption (Method II) to reinforce the NFC hydrogels. The charge density of the NFC, the incorporated hemicellulose type and amount, and the swelling time of the hydrogels were found to affect the pore structure, the mechanical strength, and thus the cells' growth on the composite hydrogel scaffolds. The XG showed the highest adsorption capacity on the NFC, the highest reinforcement effect, and facilitated/promoted cell growth. The pre-sorbed XG in the low-charged NFC network with a lower weight ratio (NFC/XG-90:10) showed the highest efficacy in supporting the growth and proliferation of fibroblast cells (NIH 3T3). These all-polysaccharide composite hydrogels may work as promising scaffolds in wound healing applications to provide supporting networks and to promote cells adhesion, growth, and proliferation. [ABSTRACT FROM AUTHOR]

Interactions among cellulose, hemicellulose and pectins are important for plant cell wall assembly and properties and also for industrial applications of these polysaccharides. Therefore, binding of pectin and xyloglucan on microcrystalline cellulose was investigated in this experiment by adsorption isotherms, zeta potential and scanning electron microscopy (SEM). Analysis of three isotherm models (Langmuir, Freundlich and Fowler-Guggenheim isotherms) showed that the experimental adsorption isotherm was well described via the Fowler-Guggenheim model, which includes lateral interaction between the adsorbate. The adsorption isotherm and zeta potential measurement showed that at temperature 25 °C only xyloglucan adsorbed on the microcrystalline cellulose. In case of xyloglucan on cellulose, the equilibrium was reached in about 3-4 h, and the kinetics of adsorption were well described by the multiexponential equation. Analysis of the model suggests that two steps can be distinguished: diffusion and reconformation in an adsorbed layer. No adsorption of pectin was observed in this study. SEM study showed that xyloglucan may prevent cellulose from aggregation. [ABSTRACT FROM AUTHOR]

Main conclusion: Xyloglucan endo-transglycosylase/hydrolase ( Ph XET/H) regulates Podophyllum seed germination via GA mediated up-accumulation of Ph XET protein and subsequent endosperm weakening. Abstract: Xyloglucan endo-transglycosylase/hydrolase (XET/H) belong to glycosyl hydrolase family 16, which play an important role in endosperm weakening and embryonic expansion during seed germination. Podophyllum hexandrum is a high altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. During seed germination in Podophyllum, accumulation of XET/H transcripts was recorded. This data confirmed its possible role in determining the fate of seed for germination. Full length cDNA of a membrane bound XET/H (here onwards PhXET) was cloned from the germinating seeds of Podophyllum. Analysis of nucleotide sequence revealed PhXET with an open reading frame of 720 bp encoding a protein of 239 amino acids with a molecular mass of 28 kDa and pI of 7.58. In silico structure prediction of PhXET showed homology with that of Populus tremula (1UN1). PhXET was predicted to have a potential GPI-anchor domain and was located in plasma membrane. It was found that the exogenously applied phytohormones (GA and ABA) regulate the expression of PhXET. The obtained data showed that the PhXET regulates seed germination in Podophyllum by supplementing its activity along with other endosperm weakening and embryo expansion genes. [ABSTRACT FROM AUTHOR]

The xyloglucan endotransglycosylase gene RcXET of China Rose ( Rosa chinensis Jacq.) and the MtDREB1C gene of Medicago truncatula Gaertn. were pyramided into the plant expression vector pBin438 and transformed into China Rose. Southern blot and Northern blot analyses showed that the heterologous gene MtDREB1C was integrated into the genome of surviving transgenic rose plants and expressed at different levels. Real-time PCR analysis demonstrated that robust expression of the congenetic gene RcXET was activated in the five surviving transgenic rose plants. The performance of the five transgenic lines under freezing and drought stress was superior to that of non-transformed controls. Thus, pyramiding of the genes MtDREB1C and RcXET in China Rose was more effective to enhance freezing and drought tolerance than untransformed controls. A positive correlation was observed between the expression of RcXET and the growth rate in contrast to the non-transgenic plants. The physiological assay showed that co-expression had greater effects on EC%, contents of proline, soluble sugar, photosynthesis rate, negative water potential, and turgor loss point than activities of POD and SOD under stress. The study also highlights the utility of a simple and rapid approach to express two or even more genes in one expression vector. [ABSTRACT FROM AUTHOR]

Main conclusion: Chemical analyses and glycome profiling demonstrate differences in the structures of the xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I isolated from soybean ( Glycine max ) roots and root hair cell walls. The root hair is a plant cell that extends only at its tip. All other root cells have the ability to grow in different directions (diffuse growth). Although both growth modes require controlled expansion of the cell wall, the types and structures of polysaccharides in the walls of diffuse and tip-growing cells from the same plant have not been determined. Soybean ( Glycine max) is one of the few plants whose root hairs can be isolated in amounts sufficient for cell wall chemical characterization. Here, we describe the structural features of rhamnogalacturonan I, rhamnogalacturonan II, xyloglucan, glucomannan, and 4- O-methyl glucuronoxylan present in the cell walls of soybean root hairs and roots stripped of root hairs. Irrespective of cell type, rhamnogalacturonan II exists as a dimer that is cross-linked by a borate ester. Root hair rhamnogalacturonan I contains more neutral oligosaccharide side chains than its root counterpart. At least 90 % of the glucuronic acid is 4- O-methylated in root glucuronoxylan. Only 50 % of this glycose is 4- O-methylated in the root hair counterpart. Mono O-acetylated fucose-containing subunits account for at least 60 % of the neutral xyloglucan from root and root hair walls. By contrast, a galacturonic acid-containing xyloglucan was detected only in root hair cell walls. Soybean homologs of the Arabidopsis xyloglucan-specific galacturonosyltransferase are highly expressed only in root hairs. A mannose-rich polysaccharide was also detected only in root hair cell walls. Our data demonstrate that the walls of tip-growing root hairs cells have structural features that distinguish them from the walls of other roots cells. [ABSTRACT FROM AUTHOR]

Tamarind seed xyloglucan (TSX) and к-carrageenan (KC) have been widely used in the food and pharmaceutical industries. Their synergistic effects and the gelation and degelation of the blends, and the release a hydrophilic model drug, nicotinamide, from the blends were investigated. The KC/TSX blend with a ratio of 1 % (w/v) of KC to 1 % (w/v) of TSX (1KC/1TSX) exhibited the highest synergistic effect. The sol-gel and gel-sol transition temperatures of the blends shifted to higher temperatures when compared with KC alone. The interactions between KC and TSX in the gel state were revealed by ATR-FTIR showing the significant differences in the position and intensity of the peaks at 1164, 1124 and 1130 cm for the 1KC/1TSX gel sample. These were probably due to the intermolecular hydrogen bonds between the OH and C-O-C groups of KC and TSX. In addition, the sol and gel states of the KC or KC/TSX blends could be differentiated using ATR-FTIR. Furthermore, the time needed to release 90 % ( T) of nicotinamide alone and nicotinamide from 2KC, 1.5KC/0.5TSX, 1KC/1TSX and 0.5KC/1.5TSX was 17, 66, 75, 95 and 78 min, respectively. This demonstrated a sustained release of the drug when incorporated into the KC or KC/TSX blends in the freeze-dried samples. Moreover, the blends retarded the release better than KC alone. The blend of 1KC/1TSX, that exhibited the highest synergism, provided the most effective retardation of the compounds. [ABSTRACT FROM AUTHOR]

Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k (chimera, 177.03 s; XegA, 144.31 s) and reduced K (chimera, 1.30 mg mL; XegA, 1.50 mg mL) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera. [ABSTRACT FROM AUTHOR]

In the present work, bacterial cellulose nanocrystals (BCN) were prepared with three distinct sulfate contents, namely 0, 0.42 and 0.65 % OSO, by using HCl (BCN-HCl), a mixture of HCl and HSO (BCN-HCl/HSO) and HSO (BCN-HSO) as hydrolytic media, respectively. BCN were characterized by means of elemental analysis, X-ray diffraction, atomic force microscopy (AFM) and zeta potential measurements. All BCN samples had crystallinity indices of about 82 %, about 10 % higher than the original BC. The zeta potential values determined for BCN-HCl, BCN-HCl/HSO and BCN-HSO amounted to −(5 ± 1), −(40 ± 1) and −(46 ± 1) mV, respectively. The assembly between BCN-HCl, BCN-HCl/HSO or BCN-HSO and XG extracted from Tamarindus indica seeds was investigated by means of Freundlich isotherms, AFM, contact angle measurements, dynamic light scattering, ellipsometry and quartz crystal microbalance measurements. The adsorption experiments suggest that sulfate substitution on BCN surface as low as 0.65 % OSO impairs the original interaction with XG, thereby generating unstable layers with subsequent desorption. In contrast, the 0.42 % OSO samples presented favorable interactions with XG and exhibited highly improved colloidal stability compared to 0 % OSO samples. Films of XG adsorbed onto BCN with 0.42 % OSO were stable, homogeneous, and had low roughness and thickness. This is the first report showing that sulfate content on BCN surface can be tailored to promote high colloidal stability and sufficient interaction with XG. [ABSTRACT FROM AUTHOR]

Main conclusion: Putative XyG xylosyltransferases from Tropaeolum majus (nasturtium) and Solanum lycopersicum (tomato) homologous to characterized Arabidopsis genes were identified and shown to functionally complement Arabidopsis mutants lacking xyloglucan demonstrating they represent xyloglucan xylosyltransferases. Xyloglucan is a major hemicellulose in the plant cell wall and is important for the structural organization of the wall. The fine structure of xyloglucan can vary dependent on plant species and tissue type. Most vascular seed-bearing plants including Arabidopsis thaliana and nasturtium ( Tropaeolum majus) have a xyloglucan structure, in which three out of four backbone glucosyl-residues are substituted with xylosyl-residues. In contrast, the xyloglucan found in plants of the Solanaceae family, which includes tomato ( Solanum lycopersicum), is typically less xylosylated with only two of the four backbone glucosyl-residues substituted with xylosyl-residues. To investigate the genetics of xyloglucan xylosylation, candidate xyloglucan xylosyltransferase genes ( XXTs) homologous to known A. thaliana XXTs were cloned from nasturtium and tomato. These candidate XXTs were expressed in the A. thaliana xxt1/2 double and xxt1/2/5 triple mutant, whose walls lack detectable xyloglucan. Expression of the orthologs of XXT5 resulted in no detectable xyloglucan in the transgenic A. thaliana plants, consistent with a lack of xyloglucan in the A. thaliana xxt1/2 double mutant. However, transformation of both the tomato and nasturtium orthologs of AtXXT1 and AtXXT2 resulted in the production of xyloglucan with a xylosylation pattern similar to wild type A. thaliana indicating that both SlXXT2 and TmXXT2 likely have xylosyltransferase activity. As the expression of the SlXXT2 did not result in xyloglucan with a decreased xylosylation frequency found in tomato, this gene is not responsible for the unique xylosylation pattern found in the solanaceous plants. [ABSTRACT FROM AUTHOR]

Computational modeling reveals that a cell wall network whose mechanical integrity is dominated by a small mass fraction of 'hotspot' linkers between microfibrils can sit close to a percolation threshold, across which mechanical integrity is very sensitive to the number of hotspots. In the model, the mechanical properties of cell wall fragments consisting of cellulose microfibrils and xyloglucan linkers with different levels of disorder were examined under progressive decimation of the network, modeling enzymatic degradation. The percolation limit so obtained is close to mass fraction of xyloglucan that must be removed to induce creep experimentally. Greater disorder in the interconnectivity of the network raises the number of hotspot linkers per fibril necessary to reach the percolation threshold. To maintain the required mechanical stiffness with a sparse network of hotspot connections, either each xyloglucan linker must be much stiffer than a single polymeric strand or an additional cell wall component, i.e. pectin, must carry substantial load with a sensitive non-linear mechanical response, such as that associated with a glass transition. [ABSTRACT FROM AUTHOR]

A xyloglucan-specific endo-1,4β-glucanase (XcXGHA) from Xanthomonas citri pv. mangiferaeindicae has been cloned, expressed in Escherichia coli, purified and characterised. The XcXGHA enzyme belongs to CAZy family GH74 and has catalytic site residues conserved with other xyloglucanases in this family. At its optimal reaction conditions, pH 7.0 and 40 °C, the enzyme has a k/ K value of 2.2 × 10 min M on a tamarind seed xyloglucan substrate. XcXGHA is relatively stable within a broad pH range (pH 4-9) and up to 50 °C ( t of 74 min). XcXGHA is proven to be xyloglucan-specific, and a glycan microarray study verifies that XcXGHA catalyses cleavage of xyloglucan extracted from both monocot and dicot plant species. The enzyme catalyses hydrolysis of tamarind xyloglucan in a unique way by cleaving XXXG into XX and XG (X is xylosyl-substituted glucose; G is unsubstituted glucose), is able to degrade more complex xyloglucans and notably is able to cleave near more substituted xyloglucan motifs such as L [i.e. α- l-Fuc p-(1 → 2)-β- d-Gal p-(1 → 2)-α- d-Xyl p-(1 → 6)-β- d-Glc p]. LC-MS/MS analysis of product profiles of tamarind xyloglucan which had been catalytically degraded by XcXGHA revealed that XcXGHA has specificity for X in subsite −1. The 3D model suggests that XcXGHA consists of two seven-bladed β-propeller domains with the catalytic center formed by the interface of these two domains, which is conserved in xyloglucanases in the GH74 family. However, the XcXGHA has two amino acids (D264 and R472) that differ from the conserved residues of other GH74 xyloglucanases. These two amino acids were predicted to be located on the opposite side of the active site pocket, facing each other and forming a closing surface above the active site pocket. These two amino acids may contribute to the unique substrate specificity of the XcXGHA enzyme. [ABSTRACT FROM AUTHOR]

The goal of this study is to investigate the hydration and dehydration induced structural changes of native cellulose. Never dried cotton, and never dried bacterial cellulose with and without added matrix polymer xyloglucan, are examined under the influence of dehydration and rehydration. Significant crystal structure changes were observed in the later stage of drying for both cotton and bacterial cellulose (BC). The 1 % lateral expansion in glucan chain spacing and 17 % decrease of calculated Scherrer dimension were detected for cotton due to the distortion of the structure possibly caused by mechanical stresses associated with drying. No detectable changes on average glucan chain spacings were observed for large BC crystals. However, an average width decrease by 4.4 nm was discovered in the (010) direction, which was more significant than that observed in the (100) and (110) directions. It is hypothesized that co-crystallized elementary fibrils preferentially disassociate along the (010) plane resulting in a significant reduction of crystal width. In the BC-xyloglucan model composite, the presence of xyloglucan does not interfere with the dehydration behavior. Rehydration leads to some structural changes but to a lesser extent than the initial drying. High temperature dehydration induced deformation and crystal size changes are found to be non-reversible due to the removal of the last hydration layer on the cellulose surface. [ABSTRACT FROM AUTHOR]

Gluconacetobacter xylinus has the ability to produce different types of water soluble exopolysaccharides (EPS). Those EPS have different levels of association to bacterial cellulose (BC). At least a portion of the EPS can be released from the BC by 0.1 to 4 M NaOH solution treatments. Hard to extract EPS (HE-EPS) released by 4 M NaOH solutions have been characterized and contain approximately 75 % mannose and 25 % glucose. To study the effect of the EPS on BC synthesis, purified EPS were added to the medium at the start of cultivation and the BC produced was characterized. Results showed that the presence of HE-EPS in the culture medium interfered with the alignment of the BC crystals, but did not reduce crystal size. This is in contrast to similar studies performed using xyloglucan, xylan and glucomannan. The width of the average ribbon increased by 60 % when HE-EPS levels increased in the medium, which indicated that the HE-EPS could also modulate the bundling of cellulose ribbons. Based on the data we propose a mechanism for how HE-EPS alters cellulose formation and assembly. The addition of HE-EPS disturbs the preferential crystal orientation and increases the spacing of cellulose microfibrils without affecting crystallization by associating with ordered cellulose prior to physical aggregation or bundling. [ABSTRACT FROM AUTHOR]

The cell wall is a dynamic structure that can be modified by different conditions during growth. Stem inclination induces biochemical changes and chemical modifications in the cell walls of conifers driving plant stem reorientation. Different enzymes take part in cell wall remodeling, including xyloglucan endotransglycosylase/hydrolase (XTH). This enzyme modifies xyloglucan, the main hemicellulose found in primary cell walls of pine trees. The full-length complementary DNA of PrXTH1 was isolated and characterized from radiata pine. PrXTH1 contains an open-reading frame of 882 bp encoding a protein of 294 amino acids, including an N-terminal signal peptide for secretion that targets the protein to the cell wall. Phylogenetic and gene structure analyses suggest that PrXTH1 belongs to group I of XTHs. The recombinant purified protein has only transglycosylase activity. A 3D model of PrXTH1 protein was built through comparative modeling, showing a β-sandwich structure with the catalytic site in the middle of the protein structure, composed of residues Glu79, Asp81, and Glu83. One-year-old half-sib seedlings were inclined at 45° and transcriptional analysis indicated that PrXTH1 transcripts are more abundant in the lower side of inclined stems. Additionally, along the stem, PrXTH1 transcript accumulation starts at the apex and moves down over time to the stem base. This gene shows regulation by ethylene, indicating that it could be a target in the hormonal cascade during the inclination response. Finally, in radiata pine seedlings PrXTH1 is induced during the inclination response, suggesting an active participation of this enzyme in the cell wall reorganization required during this phenomenon. [ABSTRACT FROM AUTHOR]

Development of cambium and its activity is important for our knowledge of the mechanism of secondary growth. Arabidopsis thaliana emerges as a good model plant for such a kind of study. Thus, this paper reports on cellular events taking place in the interfascicular regions of inflorescence stems of A. thaliana, leading to the development of interfascicular cambium from differentiated interfascicular parenchyma cells (IPC). These events are as follows: appearance of auxin accumulation, PIN1 gene expression, polar PIN1 protein localization in the basal plasma membrane and periclinal divisions. Distribution of auxin was observed to be higher in differentiating into cambium parenchyma cells compared to cells within the pith and cortex. Expression of PIN1 in IPC was always preceded by auxin accumulation. Basal localization of PIN1 was already established in the cells prior to their periclinal division. These cellular events initiated within parenchyma cells adjacent to the vascular bundles and successively extended from that point towards the middle region of the interfascicular area, located between neighboring vascular bundles. The final consequence of which was the closure of the cambial ring within the stem. Changes in the chemical composition of IPC walls were also detected and included changes of pectic epitopes, xyloglucans (XG) and extensins rich in hydroxyproline (HRGPs). In summary, results presented in this paper describe interfascicular cambium ontogenesis in terms of successive cellular events in the interfascicular regions of inflorescence stems of Arabidopsis. [ABSTRACT FROM AUTHOR]

Key message: We isolated differentially expressed and dark-responsive genes during flower development and opening in petals of morning glory. Abstract: Flower opening usually depends on petal expansion and is regulated by both genetic and environmental factors. Flower opening in morning glory ( Ipomoea nil) is controlled by the dark/light regime just prior to opening. Opening was normal after 8- or 12-h dark periods but progressed very slowly after a 4-h dark period or in continuous light. Four genes ( InXTH1- InXTH4) encoding xyloglucan endotransglucosylase/hydrolases (XTHs) and three genes ( InEXPA1- InEXPA3) encoding alpha-expansins (EXPAs) were isolated. The expression patterns of InXTH2, InXTH3, and InXTH4 in petals were closely correlated with the rate of flower opening controlled by the length of the dark period prior to opening, but those of the EXPA genes were not. The expression pattern of InXTH1 gene was closely correlated with petal elongation. Suppression subtractive hybridization was used to isolate dark-responsive genes accompanying flower opening. The expressions of ten isolated genes were associated with the length of the dark period prior to flower opening. One gene was highly homologous to Arabidopsis PSEUDO- RESPONSE REGULATOR7, which is associated with the circadian clock and phytochrome signaling; another to Arabidopsis REVEILLE1, which affects the output of the circadian clock. Other genes were related to light responses, plant hormone effects and signal transduction. The possible roles of these genes in regulation of flower opening are discussed. [ABSTRACT FROM AUTHOR]

The effect of surface hydrophobicity and side-chain variation on xyloglucan adsorption onto cellulose microfibrils (CMF) is investigated via molecular dynamics simulations. A molecular model of CMF with (100), (010), (1-10), (110) and (200) crystal faces was built. We considered xylogluco-oligosaccharides (XGO) with three repeating units, namely (XXXG) (XXLG), and (XXFG) (where each (1,4)-β- d-glucosyl residue in the backbone is given a one-letter code according to its substituents: G = β- d-Glc; X = α- d-Xyl-(1,6)-β- d-Glc; L = β- d-Gal-(1,2)-α- d-Xyl-(1,6)-β- d-Glc; F = α- l-Fuc-(1,2)-β- d-Gal-(1,2)-α- d-Xyl-(1,6)-β- d-Glc). Our work shows that (XXXG) binds more favorably to the CMF (100) and (200) hydrophobic surfaces than to the (110), (010) and (1-10) hydrophilic surfaces. The origin of this behavior is attributed to the topography of hydrophobic CMF surface, which stabilizes (XXXG) in flat conformation. In contrast, on the rough hydrophilic CMF surface (XXXG) adopts a less favorable random-coil conformation to facilitate more hydrogen bonds with the surface. Extending the xyloglucan side chains from (XXXG) to (XXLG) hinders their stacking on the CMF hydrophobic surface. For (XXFG), the interaction with the hydrophobic surface is as strong as (XXXG). All three XGOs have similar binding to the hydrophilic surface. Steered molecular dynamics simulation was performed on an adhesive model where (XXXG) was sandwiched between two CMF hydrophobic surfaces. Our analysis suggests that this sandwich structure might help provide mechanical strength for plant cell walls. Our study relates to a recently revised model of primary cell walls in which extensibility is largely determined by xyloglucan located in limited regions of tight contact between CMFs. [ABSTRACT FROM AUTHOR]

A well-balanced human diet includes a significant intake of non-starch polysaccharides, collectively termed 'dietary fibre', from the cell walls of diverse fruits and vegetables. Owing to the paucity of alimentary enzymes encoded by the human genome, our ability to derive energy from dietary fibre depends on the saccharification and fermentation of complex carbohydrates by the massive microbial community residing in our distal gut. The xyloglucans (XyGs) are a ubiquitous family of highly branched plant cell wall polysaccharides whose mechanism(s) of degradation in the human gut and consequent importance in nutrition have been unclear. Here we demonstrate that a single, complex gene locus in Bacteroides ovatus confers XyG catabolism in this common colonic symbiont. Through targeted gene disruption, biochemical analysis of all predicted glycoside hydrolases and carbohydrate-binding proteins, and three-dimensional structural determination of the vanguard endo-xyloglucanase, we reveal the molecular mechanisms through which XyGs are hydrolysed to component monosaccharides for further metabolism. We also observe that orthologous XyG utilization loci (XyGULs) serve as genetic markers of XyG catabolism in Bacteroidetes, that XyGULs are restricted to a limited number of phylogenetically diverse strains, and that XyGULs are ubiquitous in surveyed human metagenomes. Our findings reveal that the metabolism of even highly abundant components of dietary fibre may be mediated by niche species, which has immediate fundamental and practical implications for gut symbiont population ecology in the context of human diet, nutrition and health. [ABSTRACT FROM AUTHOR]

Xyloglucan and pectin are major non-cellulosic components of most primary plant cell walls. It is believed that xyloglucan and perhaps pectin are functioning as tethers between cellulose microfibrils in the cell walls. In order to understand the role of xyloglucan and pectin in cell wall mechanical properties, model cell wall composites created using Gluconacetobacter xylinus cellulose or cellulose nanowhiskers (CNWs) derived there from with different amounts of xyloglucan and/or pectin have been prepared and measured under extension conditions. Compared with pure CNW films, CNW composites with lower amounts of xyloglucan or pectin did not show significant differences in mechanical behavior. Only when the additives were as high as 60 %, the films exhibited a slightly lower Young's modulus. However, when cultured with xyloglucan or pectin, the bacterial cellulose (BC) composites produced by G. xylinus showed much lower modulus compared with that of the pure BC films. Xyloglucan was able to further reduce the modulus and extensibility of the film compared to that of pectin. It is proposed that surface coating or tethering of xyloglucan or pectin of cellulose microfibrils does not alone affect the mechanical properties of cell wall materials. The implication from this work is that xyloglucan or pectin alters the mechanical properties of cellulose networks during rather than after the cellulose biosynthesis process, which impacts the nature of the connection between these compounds. [ABSTRACT FROM AUTHOR]

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH 6.5 and 65 °C, RmXEG12B at pH 5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U mg −1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β- d-Glc residue, X is a xylosylated β- d-Glc residue, and L is a β- d-Glc residue substituted by xylosyl-galactose). [ABSTRACT FROM AUTHOR]

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH 6.5 and 65 °C, RmXEG12B at pH 5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U mg −1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β- d-Glc residue, X is a xylosylated β- d-Glc residue, and L is a β- d-Glc residue substituted by xylosyl-galactose). [ABSTRACT FROM AUTHOR]

One major component of plant cell walls is a diverse group of polysaccharides, the hemicelluloses. Hemicelluloses constitute roughly one-third of the wall biomass and encompass the heteromannans, xyloglucan, heteroxylans, and mixed-linkage glucan. The fine structure of these polysaccharides, particularly their substitution, varies depending on the plant species and tissue type. The hemicelluloses are used in numerous industrial applications such as food additives as well as in medicinal applications. Their abundance in lignocellulosic feedstocks should not be overlooked, if the utilization of this renewable resource for fuels and other commodity chemicals becomes a reality. Fortunately, our understanding of the biosynthesis of the various hemicelluloses in the plant has increased enormously in recent years mainly through genetic approaches. Taking advantage of this knowledge has led to plant mutants with altered hemicellulosic structures demonstrating the importance of the hemicelluloses in plant growth and development. However, while we are on a solid trajectory in identifying all necessary genes/proteins involved in hemicellulose biosynthesis, future research is required to combine these single components and assemble them to gain a holistic mechanistic understanding of the biosynthesis of this important class of plant cell wall polysaccharides. [ABSTRACT FROM AUTHOR]

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on l-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene ( FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ∼1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily. [ABSTRACT FROM AUTHOR]

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble β-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182-191, ). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble β-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the β-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this β-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure. [ABSTRACT FROM AUTHOR]

By combining anion-exchange chromatography with gel filtration, an effective method for purification of wild-type xyloglucanase and five other cellulolytic enzymes from strain QM9414 of Trichoderma reesei was established. Characterization by enzyme activity assay, SDS-PAGE, and mass spectrometry identified the purified proteins as cellobiohydrolases I and II, endoglucanases I and II, a xyloglucanase, and β-xylosidase, of which the xyloglucanase was purified for the first time from the mutant strain QM9414. This method holds great promise to study the mechanism of cellulolytic enzymes, to investigate the synergistic action between cellulase and other cellulolytic enzymes, and to better exploit enzyme preparations for degradation of lignocellulose. [ABSTRACT FROM AUTHOR]

This is the first report of inactivation of xyloglucanase from Thermomonospora sp by pepstatin A, a specific inhibitor towards aspartic proteases. The steady state kinetics revealed a reversible, competitive, two-step inhibition mechanism with IC and K values of 3.5 ± 0.5 μM and 1.25 ± 0.5 μM respectively. The rate constants determined for the isomerization of EI to EI and the dissociation of EI* were 14.5 ± 1.5 × 10 s and 2.85 ± 1.2 × 10 s respectively, whereas the overall inhibition constant K was 27 ± 1 nM. The conformational changes induced upon inhibitor binding to xyloglucanase were monitored by fluorescence analysis and the rate constants derived were in agreement with the kinetic data. The abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled xyloglucanase and far UV analysis suggested that pepstatin binds to the active site of the enzyme. Our results revealed that the inactivation of xyloglucanase is due to the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis. [ABSTRACT FROM AUTHOR]

Proteinaceous inhibitors of plant cell wall degrading enzymes can provide a first level of defense from invasive phytopathogens. One recently discovered inhibitor is the xyloglucans-specific endoglucanase inhibitor protein (XEGIP) originally found in tomato. This inhibitor protein has since been found in many solanaceous plants as well as other dicots. As xyloglucans play a major role in maintaining cell wall structure in dicots, protection of xyloglucan is critical. Thus far, only a single potato XEGIP had been identified (AY321357), even though a major pathogen of potato, Phytophthora infestans, has multiple copies of xyloglucan-specific endoglucanase. We now report that potato has nine additional copies of XEGIP, clustered together on a small region of chromosome one. They are located adjacent to the previously described potato XEGIP and represent one of the highest degrees of gene duplication relative to any other potato inhibitor. Synteny with tomato indicates this duplication may have occurred before speciation of Solanum. [ABSTRACT FROM AUTHOR]

The localization of cell wall polysaccharides of the fused petals of monocotyledonous Sandersonia aurantiaca flowers has been identified using antibodies directed to pectin and xyloglucan epitopes and detection by fluorescence microscopy. Cross sections of the petal tissue were taken from cut flowers in bud and at various stages of maturity and senescence. Patterns of esterification in pectin backbones were identified by JIM5 and 2F4 labelling. Pectic galactan and arabinan side branches were detected by LM5 and LM6, respectively, while fucosylated xyloglucan was identified by CCRC-M1. The labelling patterns highlighted compositional differences between walls of the outer/inner epidermis compared to the spongy parenchyma cells of the interior mesophyll for fucosylated xyloglucan and arabinan. Partially esterified homogalacturonan was present in the junction zones of the outer epidermis and points of contact between cells of the mesophyll, and persisted throughout senescence. Pectic galactans were ubiquitous in the outer and inner epidermal cell walls and walls of the interior mesophyll at flower opening, whereas pectic arabinan was found predominantly in the epidermal cells. Galactan was lost from walls of all cells as flowers began to senesce, while fucosylated xyloglucan appeared to increase over this time. Such differences in the location of polysaccharides and the timing of changes suggest distinct combinations of certain polysaccharides offer mechanical and rheological advantages that may assist with flower opening and senescence. [ABSTRACT FROM AUTHOR]