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A series of oligomeric glycans can be extracted from the cell walls of developing cotton fibers with weak acid. Glycans that produce similar profiles on high pH anion chromatography with pulsed amperometric detection (HPAEC-PAD) are also found in a protein complex extracted from developing fibers and in amorphous aggregates found in association with immature fibers in developing, but not in mature cotton bolls. The quantity and composition of the glycans recovered from the carbohydrate-protein complex varies significantly with the time of day when the bolls are harvested. This diurnal variation is consistent with the hypothesis that secondary cell walls are deposited primarily at night. Incubation of re-hydrated cotton fibers in the presence of exogenous oligosaccharides, myo-inositol and glycerol substantially alters the apparent quantity of the oligomers extracted from the fibers. The same and similar glycans have also been extracted from cotton fabric, marine algae, various paper products and wood. While many of the oligomers isolated from the various cellulose sources display the same peaks by HPAEC-PAD, the specific number of oligomers and their relative quantities appear unique for each source of cellulosic material. Oligomeric glycans, as described in the preceding, are present in all cellulose sources that have been investigated. Their relative abundance changes in response to source, stage of development and other physiological variables. We hypothesize that the glycans are intermediates in the biological assembly of cellulose, and that their incorporation in cellulose is mediated by physicochemical and enzymatic mechanisms.

A cDNA encoding a stilbene synthase, RtSTS, was isolated from the rhizomes of Tatar rhubarb, Rheum tataricum L. (Polygonaceae), a medicinal plant containing stilbenes and other polyketides. Recombinant RtSTS was expressed in E. coli and assayed with acetyl-coenzyme A (CoA), n-butyryl-CoA, isovaleryl-CoA, n-hexanoyl-CoA, cinnamoyl-CoA and p-coumaroyl-CoA as primers of polyketide synthesis. RtSTS synthesized resveratrol and a trace amount of naringenin chalcone from p-coumaroyl-CoA, supporting the enzyme's identification as a resveratrol-type stilbene synthase (EC 2.3.1.95). Bis-noryangonin and p-coumaroyl triacetic acid lactone (CTAL)-type pyrones were observed in minor amounts in the reaction with p-coumaroyl-CoA and as major products with cinnamoyl CoA. As well, such pyrones, and not aromatic polyketides, were identified as the only products in assays with aliphatic and benzoyl CoA esters. Acetonyl-4-hydroxy-2-pyrone, a pyrone synthesized from acetyl-CoA, was identified as a new product of a stilbene synthase. Using Northern blot analysis, RtSTS transcript was found to be highly expressed in R. tataricum rhizomes, with low transcript levels also present in young leaves. This expression pattern correlated with the occurrence of resveratrol, which was detected in higher amounts in R. tataricum rhizomes compared with leaves and petioles using HPLC. Few stilbene synthases have been found in plants, and the identification of RtSTS provides additional sequence and catalytic information with which to study the evolution of plant polyketide synthases.

A pair of new aryl isochroman lignin trimers, 7-acetoxy-1-{2-acetoxy-1-[4-(3-acetoxyprop-2-enyl)]-2-methoxyphenoxy}-3-(4-acetoxy-3-methoxyphenyl)-4-acetoxymethyl-6-methoxy-isochromans, along with three related dimers were isolated from DFRC degraded loblolly pine (Pinus taeda L.) wood following gel permeation and reversed-phase TLC and HPLC. Their structures were elucidated by means of 2D–NMR, FAB–MS and GC–MS analyses. These two trimers contained a unique β–1 inter-unit linkage with a 6-membered di-α-ether ring and with the original sidechain migrated. The mechanistic plausibility of the lignification pathway, the isolation of expected DFRC degradation dimers and trimers, and the tentative identification of the aryl isochroman structure in pine milled wood lignins by NMR spectroscopy, all suggest that such structures, or their precursors, are present in native lignins (in their unacetylated forms). An unrelated trimer possessing 5–5/β–β interunit linkages was also isolated and characterized.

The stereostructures and molecular conformations of four more diterpene lactones, gelomulides G-J, isolated as minor constituents from the leaves of Gelonium multiflorum were established on the basis of their 1H NMR, 13C NMR and mass spectral evidence. Additionally, a 2D NMR spectral study and the ORTEP diagram derived from the X-ray crystallographic analysis of gelomulide I confirmed its proposed stereostructure and absolute configuration.

A β-glucosidase preparation from 'Marianna' plum was characterized. Only monoglycosides with a β-linkage to aromatic aglycones were hydrolyzed. A single pH optimum at 5·3 was found. The highest specific activity occurred with the substrate prunasin (d(—)-mandelonitrile-β-glucopyranoside). β-Galactosidase activity was also detected.

the R f values of twenty-two carotenoids separated on Whatman paper Chromedia AH 81 and SG81 are given. The R f values are readily reproducible and separation of a large number of carotenoids can easily be carried out in about half-an-hour.

Clausena lansium (Lour.) Skeels (Rutaceae) is a natural bioactive plant. Its roots, stems, leaves, and seeds are widely used in Chinese traditional and folk medicine. Although the characterization and functional analysis of bioactive components in Clausena lansium (Lour.) Skeels has been widely reported, the spatial distribution of these compounds within the main plant tissues remains undefined. Here, we adopted matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to reveal the spatial distribution of active alkaloids, coumarins, sugars and organic acids in C. lansium. Using a combined wet and dry matrix covering method to enhance sensitivity, we detected alkaloids throughout the fruit including 3-methylcarbazole and murrastinine which were especially rich in the kernel tissues but were restricted to the stem xylem and medulla and in the leaf epidermal region. Interestingly, murrayanine and heptaphylline were mainly found in pulp tissues with very low content in the stems and leaves while girinimbine was only distributed within the outer kernel skin. Coumarins were mainly distributed in the fruit pericarp and leaf vein tissues but with no clear spatial specificity in stems. Lastly, hexoses were mainly evident in the fruit pulp, although sucrose was also found in the pericarp, pulp, and pulp fibers with citric acid being distributed throughout the fruit. The accurate spatial and chemical information obtained provides new insights into the specific accumulation of metabolites in individual tissues.

Driselase-digestion of cell walls from suspension-cultures of spinach (Spinacia oleracea L.), followed by anion-exchange chromatography, gel-permeation chromatography, preparative paper chromatography and preparative paper electrophoresis, yielded ten uronic acid-containing products in addition to free galacturonic acid (GalA). These included 4-O-methylglucuronic acid, alpha-L-rhamnopyranosyl-(1-->4)-D-glucuronic acid and several oligosaccharides containing GalA residues. The structures were unambiguously determined by a combination of 1- and 2-dimensional NMR spectroscopic techniques. Five of the six homogalacturonan-derived oligosaccharides purified contained 3-O-acetyl-GalA residues; however, methyl-esterified GalA residues occurred adjacent to both 2-O-acetyl-GalA and 3-O-acetyl-GalA residues. An acetylated, rhamnogalacturonan-I-derived oligosaccharide that was purified also contained 3-O-acetyl-GalA residues. Taken together with published data, our findings indicate considerable diversity in the patterns of pectin esterification. The implications for the action of pectin esterases are discussed.

Under the guidance of anti-fungal bioassay, four previously undescribed oleanane-type and one lupane-type triterpenoid saponins, along with twelve known analogues, were isolated from the extract of Sapindus mukorossi pulps. Their structures were determined on the basis of spectroscopic analysis and chemical methods. In vitro biotests, oleanolic acid 3-O-β-D-xylopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside showed inhibitory activity against Trichophyton rubrum with MIC80 value of 8 μg/mL, while oleanolic acid 3-O-α-L-arabinopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside exhibited inhibitory activity against both Trichophyton rubrum and Candida albicans with MIC80 values of 8 μg/mL.

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world's supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

The new monoterpenoids (2E)-2,6-dimethyl-2,5-heptadienoic acid, (2E)-2,6-dimethyl-2,5-heptadienoic acid beta-D-glucopyranosyl ester, (5E)-2,6-dimethyl-5,7-octadiene-2,3-diol, and (3E)-3,7-dimethyl-3-octene-1,2,6,7-tetrol were isolated from the fruit pulp of Passiflora quadrangularis along with the known 2,5-dimethyl-4-hydroxy-3(2H)-furanone beta-D-glucopyranoside.

In an investigation of the peel and pulp of the fresh ripe fruits of Murraya paniculata nine flavonoids: 5,7,3′,4′,5′-pentamethoxyflavanonol, 5,6,7,3′,4′,5′-hexamethoxyflavone, 3,5,6,7,3′,4′,5′-heptamethoxyflavone, 5,7,8,3′,4′,5′-hexamethoxyflavone, 3,5,7,8,3′,4′,5′-heptamethoxyflavone, 3,5,7,8,3′,4′-hexamethoxyflavone, 5-hydroxy-3,7,8,3′,4′-pentamethoxyflavone, 5-hydroxy-3,7,8,3′,4′,5′-hexamethoxyflavone and 8-hydroxy-3,5,7,3′,4′,5′-hexamethoxyflavone, were identified. The latter two compounds appear to be novel.

Sugar-beet pulp was found to contain dehydrodimers of ferulic acid, which were characterized and quantified by GC and GC-mass spectrometry. These compounds arise from 8-5′, 5-5′, 8-8′ and 8-O-4′ coupling, the 8-5′ form being preponderant; no 4-O-5′ dimer was detected. Total dehydrodimers represented 0.14% (w/w) of the pulp. Their release was attempted by treatment of the pulp with a commercial pectinase. However, diferulates were resistant to this enzymic hydrolysis; free diferulic acids were not released and remained esterified to residual structures of the sugar-beet pectins.

Extracellular polysaccharide produced by suspension-cultured Mentha cells consisted of 50% neutral sugars, 32% uronic acid and 10% of protein. The ammonium oxalate-soluble fraction of this polysaccharide contained 30% hemicellulose and 70% pectic substances. The purified hemicellulose contained xylose, glucose, arabinose, galactose, mannose and fucose residues in a molar ratio of 41.6: 31.3: 13.1: 11.1: 1.3: 1.6. It was identified as a xyloglucan from its neutral sugar composition and by methylation analysis and cellulase treatment: the principal neutral sugar was arabinose. The presence of O -acetyl residues was confirmed with 1 H NMR, 13 C NMR and GC-mass spectrometry. The total acetyl content in the polysaccharide was 4%. The point of attachment of the O -acetyl residue was shown to be at position 6 of the galactosyl residue.

Lignin was extracted from blackcurrant (Ribes nigrum) by ball-milling debarked stems in 90% dioxane. The preparation was digested with Driselase before

Two pools of hexokinase activities differing in sensitivity to ADP inhibition were characterised in maize roots. In order to evaluate how glucose utilisation could be affected by these hexokinases, glucose-6-P and NDP-5′-sugar levels were measured after a D -[U- 14 C ]glucose pulse in root extracts in the presence of 0 or 1 mM ADP. Analysis of radio-labelled activated sugars by paper chromatography revealed that: (1) without ADP, nearly 20% of the 14 C appeared in NDP-5′-sugars; (2) 0.1 mM ADP inhibited 14 C -NDP-5′-sugar formation by 85%; and (3) with 1 mM ADP, 14 C - NDP-5′-sugars were undetectable, but substantial (14%) 14 C accumulated as glucose-6-P. Mannoheptulose, a hexokinase inhibitor, blocked the NDP-5′-sugar formation, but did not modify the amount of 14 C -glucose-6-P in root extracts either with or without ADP. The analysis of the hexokinase activities with 0.8 mM glucose in maize root extracts showed that: (1) mitochondrial hexokinase activity was totally inhibited by 30 mM mannoheptulose; and (2) the cytosolic hexokinase was inhibited by only 30%. These data suggest that NDP-5′-sugar synthesis is sensitive to ADP fluctuations and that mannoheptulose affects preferentially the mitochondrial-bound hexokinase, but the cytosolic form is less sensitive. We propose that the mitochondrial hexokinase is the main energy charge sensor in this pathway in maize.

Structural elucidation of two pairs of enantiomers of 3-methoxyflavones substituted by a dihydrostilbene in the B ring is discussed using spectral evidence. These enantiomers were separated by paper chromatography.

β-Amylase from mustard ( Sinapis alba ) cotyledons was purified to homogeneity by affinity chromatography on a starch column. M r determination by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration revealed that the native enzyme is a monomer of 58 000. The analysis of products of enzyme action on amylose by paper chromatography and optical rotation revealed the exclusive formation of β-maltose as a product confirming the molecular nature of the purified enzyme as β-amylase. The substrate specificity of mustard β-amylase was akin to other plant β-amylases. The K m value for β-amylase with amylose as substrate was 0.24%. The enzyme was fairly stable at or below ambient temperature, to repeated freezing and thawing and exposure to a wide pH range (3–8). The inclusion of dithiothreitol in storage buffer improved the stability of enzyme. The enzyme was susceptible to denaturation (70–90% inactivation) by heavy metal ions (Cu 2+ , Pb 2+ , Ag + ) and sulphydryl reagents such as p -chloromercuribenzoate, indicating the sulphydryl nature of the enzyme. In contrast, α-cyclodextrin, a competitive inhibitor of β-amylase caused only a mild reduction in enzyme activity.

From the methanol extract of the fruit pulp of Lagenaria breviflora , three new saponins were characterized as 3- O -β-galactopyranosyl 28- O -β-xylopyranosyl(1–4)-α-rhamnopyranosyl(1–3)-β-xylopyranosyl(1–3)-α-arabino-pyranosylolean-12-en-28-oic acid ester, 3- O -β-galactopyranosyl 28- O -β-galactopyranosyl(1–4)-α-rhamnopyranosyl (1–3)-β-xylopyranosyl(1–3)-α-arabinopyranosylolean-12-en-28-oic acid ester and 3- O -β-galactopyranosyl 28- O -α-arabinopyranosyl(1–6)-β-galactopyranosyl(1–4)-α-rhamnopyranosyl(1–3)-β-xylopyranosyl-(1–3)-α-arabinopyranosylolean-12-en-28-oic acid ester. Oleanolic acid and 3- O -acetyloleanolic acid were identified from the hydrolytic products of the pulp.

Alcohol-insoluble residues (AIRs) from Festuca and Zea cell cultures contained 7.4 and 35 nmol esterified ferulate mg −1, respectively. Driselase solubilised 79% of the feruloylated material from both AIRs. Of the feruloyl esters solubilised from Festuca and Zea AIRs, 72 and 56% respectively were small enough to be mobile on paper chromatography. The major feruloylated product of Zea AIR was the known 5-O-feruloyl-α-l-Araf-(1 → 3)-β-d-Xylp-(1 → 4)- d-Xyl (Fer-Ara-Xyl-Xyl). In contrast, the smallest major feruloylated product of Festuca AIR was a feruloyl pentasaccharide (3) containing 3 Xyl, 1 Ara and 1 non-pentose residue (NPR). The Ara and two of the three Xyl groups of 3 were resistant to NaIO4. Mild acid hydrolysis of 3 gave xylobiose, a feruloyl trisaccharide and β-d-Xylp-(1 → 2)-(5-O- feruloyl)-l-Ara. Compound 3 was therefore NPR-(1 → 3)-β-d-Xylp-(1 → 2)-(5-O-feruloyl)-β-l--Ara f-(1 → 3)-β-d-Xylp-(1 → 4)-d-Xyl. We conclude that the complex feruloyl oligosaccharide side-chains of Festuca arabinoxylan do not protect the polysaccharide against hydrolysis by the fungal glycanases present in Driselase. © 1997 Elsevier Science Ltd. All rights reserved

The structures of the pigments in the deep purple flowers of a lisianthus line, transformed with a UDP-glucose: flavonoid-3- O -glucosyltransferase cDNA from Antirrhinum majus , have been studied using paper chromatography, HPLC and 1 H and 13 C NMR spectroscopy involving the use of 1 H 1 H COSY and TOCSY techniques. The transgenic line is shown to have produced the new anthocyanins delphinidin -3-O-β- d -(6-O-α- l - rhamnopyranosylglucopyranoside )-5-O-β- d -[6-E ( and Z)>-p- coumaroylglucopyranoside ] and delphinidin -3-O-β- d - glucopyranoside -5-O-β- d >-(6-O-E-p- coumaroylglucopyranoside ) in addition to those found in the untransformed control plants.