Ahmed Faik

A Glucurono(arabino)xylan Synthase Complex from Wheat Contains Members of the GT43, GT47, and GT75 Families and Functions Cooperatively

Glucuronoarabinoxylans (GAXs) are the major hemicelluloses in grass cell walls, but the proteins that synthesize them have previously been uncharacterized. The biosynthesis of GAXs would require at least three glycosyltransferases (GTs): xylosyltransferase (XylT), arabinosyltransferase (AraT), and glucuronosyltransferase (GlcAT). A combination of proteomics and transcriptomics analyses revealed three wheat (Triticum aestivum) glycosyltransferase (TaGT) proteins from the GT43, GT47, and GT75 families as promising candidates involved in GAX synthesis in wheat, namely TaGT43-4, TaGT47-13, and TaGT75-4. Coimmunoprecipitation experiments using specific antibodies produced against TaGT43-4 allowed the immunopurification of a complex containing these three GT proteins. The affinity-purified complex also showed GAX-XylT, GAX-AraT, and GAX-GlcAT activities that work in a cooperative manner. UDP Xyl strongly enhanced both AraT and GlcAT activities. However, while UDP arabinopyranose stimulated the XylT activity, it had only limited effect on GlcAT activity. Similarly, UDP GlcUA stimulated the XylT activity but had only limited effect on AraT activity. The [14C]GAX polymer synthesized by the affinity-purified complex contained Xyl, Ara, and GlcUA in a ratio of 45:12:1, respectively. When this product was digested with purified endoxylanase III and analyzed by high-pH anion-exchange chromatography, only two oligosaccharides were obtained, suggesting a regular structure. One of the two oligosaccharides has six Xyls and two Aras, and the second oligosaccharide contains Xyl, Ara, and GlcUA in a ratio of 40:8:1, respectively. Our results provide a direct link of the involvement of TaGT43-4, TaGT47-13, and TaGT75-4 proteins (as a core complex) in the synthesis of GAX polymer in wheat.

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO(2) (SC-CO(2)), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO(2) pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO(2) were higher than the untreated samples 12% glucose yield (12 g/100g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO(2) pretreatment at 3500 psi and 150°C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO(2) treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.

Xylan Biosynthesis: News from the Grass

Plant cell wall polysaccharides are critical components of numerous products in our everyday life. Because of this, modification of their constituent components offers unique opportunities for product improvement and economic advancement. Xylans are one of these polysaccharides that have a variety

Putative fasciclin-like arabinogalactan-proteins (FLA) in wheat (Triticum aestivum) and rice (Oryza sativa): identification and bioinformatic analyses

Putative plant adhesion molecules include arabinogalactan-proteins having fasciclin-like domains. In animal, fasciclin proteins participate in cell adhesion and communication. However, the molecular basis of interactions in plants is still unknown and none of these domains have been characterized in cereals. This work reports the characterization of 34 wheat (Triticum aestivum) and 24 rice (Oryza sativa) Fasciclin-Like Arabinogalactan-proteins (FLAs). Bioinformatics analyses show that cereal FLAs share structural characteristics with known Arabidopsis FLAs including arabinogalactan-protein and fasciclin conserved domains. At least 70% of the wheat and rice FLAs are predicted to be glycosylphosphatidylinositol-anchored to the plasma membranes. Expression analyses determined from the relative abundance of ESTs in the publicly available wheat EST databases and from RNA gel blots indicate that most of these genes are weakly expressed and found mainly in seeds and roots. Furthermore, most wheat genes were down regulated by abiotic stresses except for TaFLA9 and 12 where cold treatment induces their expression in roots. Plant fasciclin-like domains were predicted to have 3-D homology with FAS1 domain of the fasciclin I insect neural cell adhesion molecule with an estimated precision above 70%. The structural analysis shows that negatively charged amino acids are concentrated along the β1-α3-α4-β2 edges, while the positively charged amino acids are concentrated on the back side of the folds. This highly charged surface distribution could provide a way of mediating protein–protein interactions via electrostatic forces similar to many other adhesion molecules. The identification of wheat FLAs will facilitate studying their function in plant growth and development and their role in stress response.

Functional Identification of Two Nonredundant Arabidopsis α(1,2)Fucosyltransferases Specific to Arabinogalactan Proteins

Virtually nothing is known about the mechanisms and enzymes responsible for the glycosylation of arabinogalactan proteins (AGPs). The glycosyltransferase 37 family contains plant-specific enzymes, which suggests involvement in plant-specific organs such as the cell wall. Our working hypothesis is that AtFUT4 and AtFUT6 genes encode α(1,2)fucosyltransferases (FUTs) for AGPs. Multiple lines of evidence support this hypothesis. First, overexpression of the two genes in tobacco BY2 cells, known to contain nonfucosylated AGPs, resulted in a staining of transgenic cells with eel lectin, which specifically binds to terminal α-linked fucose. Second, monosaccharide analysis by high pH anion exchange chromatography and electrospray ionization mass spectrometry indicated the presence of fucose in AGPs from transgenic cell lines but not in AGPs from wild type cells. Third, detergent extracts from microsomal membranes prepared from transgenic lines were able to fucosylate, in vitro, purified AGPs from BY2 wild type cells. Susceptibility of [14C]fucosylated AGPs to α(1,2)fucosidase, and not to α(1,3/4)fucosidase, indicated that an α(1,2) linkage is formed. Furthermore, dearabinosylated AGPs were not substrate acceptors for these enzymes, indicating that arabinosyl residues represent the fucosylation sites on these molecules. Testing of several polysaccharides, oligosaccharides, and glycoproteins as potential substrate acceptors in the fucosyl transfer reactions indicated that the two enzymes are specific for AGPs but are not functionally redundant because they differentially fucosylate certain AGPs. AtFUT4 and AtFUT6 are the first enzymes to be characterized for AGP glycosylation and further our understanding of cell wall biosynthesis.

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

Background: Little is known about the enzymes involved in O-glycosylation of arabinogalactan proteins (AGPs) in plants. Results: Heterologously expressed AtGALT2 (At4g21060) catalyzed the addition of galactose to hydroxyproline in AGP peptide substrates. Conclusion: AtGALT2 is a galactosyltransferase responsible for initial galactosylation of AGPs. Significance: This work broadens our understanding of plant cell wall biosynthesis and provides an access point to identify other AGP glycosyltransferases. Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [14C]Gal from UDP-[14C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)7 and anhydrous hydrogen fluoride-deglycosylated d(AO)51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [14C]Gal to (AO)7, and the resulting product co-eluted with (AO)7 by reverse-phase HPLC. Acid hydrolysis of the [14C]Gal-(AO)7 product released 14C-radiolabel as Gal only. Base hydrolysis of the [14C]Gal-(AO)7 product released a 14C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg2+ or Mn2+ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.

UDP-Xylose-Stimulated Glucuronyltransferase Activity in Wheat Microsomal Membranes: Characterization and Role in Glucurono(arabino)xylan Biosynthesis

Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5′-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the 14C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [14C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD).

Supercritical CO2 and ionic liquids for the pretreatment of lignocellulosic biomass in bioethanol production

Owing to high petroleum prices, there has been a major push in recent years to use lignocellulosic biomass as biorefinery feedstocks. Unfortunately, by natures design, lignocellulosic biomass is notoriously recalcitrant. Cellulose is the most abundant renewable carbon source on the planet and comprises glucan polysaccharides which self-assemble into paracrystalline microfibrils. The extent of cellulose crystallinity largely contributes to biomass recalcitrance. Additionally, cellulose microfibrils are embedded into both hemicellulose and lignin polymeric networks, making cellulose accessibility an additional obstacle. Pretreatment is necessary before enzymatic hydrolysis in order to liberate high yields of glucose and other fermentable sugars from biomass polysaccharides. This work discusses two pretreatment methods, supercritical CO2 and ionic liquids (ILs). Both methods utilize green solvents that do not emit toxic vapours. Mechanisms for destroying or weakening biomass recalcitrance have been explored. Various pretreatment operating parameters such as temperature, pressure, time, dry biomass/solvent ratio, water content, etc. have been investigated for the pretreatment of various biomass types such as corn stover, switchgrass, sugarcane bagasse, soft and hard wood. The two pretreatment methods have their pros and cons. For example, supercritical CO2 explosion pretreatment uses inexpensive CO2, but requires a high pressure. By comparison, while IL pretreatment does not require an elevated pressure, ILs are still too expensive for large-scale uses. Further research and development are needed to make the two green pretreatment methods practical.

RGD‐dependent growth of maize calluses and immunodetection of an integrin‐like protein

When maize calluses are grown in the presence of the RGD peptide, important morphological changes are observed indicating the presence of a likely RGD‐binding receptor. Polyclonal antibodies generated against the human β1 integrin subunit, the platelet integrin αIIbβ3 (P23) and antibodies specific for either the β3 platelet chain or the αIIb polypeptide cross‐react with glycoproteins in Western blot analyses. Immunoprecipitation assays indicate that this maize integrin‐like protein shares structural similarities with the animal αIIbβ3 complex. We also show that AcAt2, a polyclonal antibody raised against Arabidopsis proteins purified on an RGD column, interacts with a maize protein.

Xyloglucan Galactosyl- and Fucosyltransferase Activities from Pea Epicotyl Microsomes

Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific [beta]-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs.