Biosynthesis of Alkali Insoluble Polysaccharide from UDP-d-Glucose with Particulate Enzyme Preparations from Phaseolus aureus

A particulate enzyme system from Phaseolus aureus seedlings catalyzes the synthesis of alkali insoluble polysaccharide material from UDP-d-glucose. 80 to 90% of the d-glucose units are joined by β-1,4 linkages, the remainder being combined by β-1,3 linkages. It is not known whether the material is a single polysaccharide or a mixture.     

Pathway of Uridine Diphosphate N-Acetyl-d-Glucosamine Biosynthesis in Phaseolus aureus

Studies with extracts obtained from mung beans (Phaseolus aureus) showed that UDP-N-acetyl d-glucosamine is formed from d-fructose 6-phosphate by a series of the following enzymic reactions: [Formula: see text]     

Biosynthesis of antifungal isoflavonoids in Lupinus albus. Enzymatic prenylation of genistein and 2'-hydroxygenistein.

A 45,000g particulate fraction from hypocotyls of Lupinus albus catalyzes the prenylation of genistein 1 and 2′-hydroxygenistein 2 with dimethylallyl pyrophosphate to the antifungal 6-isopentenylgenistein 3 (wighteone) and 6-isopentenyl-2′-hydroxygenistein 4 (luteone), respectively.     

The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaseolus aureus

Sucrose-phosphate synthetase is detectable only in intact chloroplast preparations of Phaseolus aureus. In contrast, sucrose synthetase and uridine diphosphate glucose (UDP-glucose) pyrophosphorylase activities are low in extracts of photosynthetic tissues of P. aureus but are high in extracts of nonphotosynthetic tissues. Activities for ADP-, dTDP-, CDP-, and GDP-glucose pyrophosphorylases are generally higher in extracts of photosynthetic tissues of P. aureus than in extracts of nonphotosynthetic tissues. The high levels of sucrose synthetase and of UDP-glucose pyrophosphorylase found in dark-grown hypocotyls begin to decline about 4 hours after exposure to light at a rate of 50% every 3 hours.     

Organogenesis from hypocotyl thin cell layers of Lupinus mutabilis and Lupinus albus

When thin cell layers (TCLn), isolated from the nodalregion of the hypocotyl of 5 day old seedlings, werecultured in medium M1 with IAA and BA, entire plantswere obtained from L. mutabilis explants andprolific shoots from L. albus explants.Histological studies carried out on L. mutabilisTCLn showed that meristematic cells, present in theexplants at the time of inoculation, gave rise to theformation of a primary meristem resulting inorganogenesis. In TCLn where apical or axillarymeristematic cells were absent, we observed theformation of a nodular structure with a cambium-likelayer at its periphery which divided to form a primarymeristem leading to organogenesis. Plant regenerationfrom TCLn may be useful in genetic transformationassays.     

Biosynthesis of galactan by a particulate enzyme preparation from Phaseolus aureus seedlings

A particulate cell-free enzyme system was prepared from Phaseolus aureus shoots. This preparation was able to incorporate [(14)C]galactose from UDP-[(14)C]galactose into a water-soluble polysaccharide, which has a probable molecular weight of at least 4600. The only labelled component detectable in the polymer was shown to be [(14)C]galactose; two labelled oligosaccharides containing only [(14)C]galactose were isolated by partial hydrolysis. The galactan-synthesizing activity of this particulate preparation is maximal at 30 degrees and pH7.1 in the presence of 5.0mm-magnesium chloride and 0.2m-sucrose. Although 3-day-old seedlings were used as a source of enzyme, it appears that 4- or 5-day-old beans contain greater synthetase activity. The enzyme system has an apparent Michaelis constant of 5.8x10(-6)m, and will catalyse the polymerization of galactose residues at the rate of 7.5mmumoles/mg. of protein/min. at a substrate concentration of 9.6mm.     

Biosynthesis, accumulation and secretion of isoflavonoids during germination and development of white lupin (Lupinus albus L.)

A combination of ¹⁴C labelling experiments of white lupin seedlings(Lupinus albus L.) and high pressure liquid chromatography oftissue extracts indicated active biosynthesis of isoflavonoidsduring the first 2–4 d of seed germination. These weresynthesized mostly as glucosides and to a lesser extent as aglycones,with trace amounts of prenylated derivatives. There was a generaldecrease in total isoflavonoids during later stages of germination(c. until d 13), which may be ascribed to their turnover and/orexudation into the rhizosphere. In addition, exudation of isoflavonoidsby lupin seeds germinated under sterile conditions continuesfor 12 d with a preferential release of monoprenylated compounds.The relationship between the specific developmental events duringseed germination and the accumulation of certain groups of isoflavonoidsis discussed in relation to their possible role in the growthprocesses of lupin. Key words: Isoflavonoids, glucosides, aglycones, prenylated derivatives, root exudates, white lupin     

The Incorporation of d-Glucosamine into Glycolipids and Glycoproteins of Membrane Preparations from Phaseolus aureus Hypocotyls

Radioactivity from d-glucosamine-(14)C is incorporated into particulate fractions of hypocotyls of Phaseolus aureus (mung bean) seedlings. Polyacrylamide gel electrophoresis of the sodium dodecyl sulfate-solubilized materials revealed that several polypeptide components varying considerably in molecular weight had become radioactive during the incubation. A considerable amount of (14)C was also recovered in lipid. Equilibrium centrifugation of the particulate material, isolated by initial centrifugation at 100,000 times gravity on sucrose density gradients revealed that radioactivity was recoverable in all of the membrane fractions along the gradient. It is suggested that glycoproteins and glycolipids containing amino sugar are normal constituents of such membranes. The ability of the particulate preparations to catalyze the transfer of N-acetyl-d-glucosamine from uridine diphospho-N-acetyl-d-glucosamine to endogenous acceptor material was also tested. Transfer was optimal at around pH 9 and in the presence of 10 mm Mg(2+), and it occurred largely into an unidentified lipid fraction. After equilibrium centrifugation of crude membrane material on sucrose gradients, a number of distinct fractions could be detected which would catalyze the transfer reaction. Uridine diphospho-d-glucose transferase activity showed a similar but not identical distribution along the gradient.     

Characterization of an isoflavonoid-specific prenyltransferase from Lupinus albus

Prenylated flavonoids and isoflavonoids possess antimicrobial activity against fungal pathogens of plants. However, only a few plant flavonoid and isoflavonoid prenyltransferase genes have been identified to date. In this study, an isoflavonoid prenyltransferase gene, designated as LaPT1, was identified from white lupin (Lupinus albus). The deduced protein sequence of LaPT1 shared high homologies with known flavonoid and isoflavonoid prenyltransferases. The LaPT1 gene was mainly expressed in roots, a major site for constitutive accumulation of prenylated isoflavones in white lupin. LaPT1 is predicted to be a membrane-bound protein with nine transmembrane regions and conserved functional domains similar to other flavonoid and isoflavonoid prenyltransferases; it has a predicted chloroplast transit peptide and is plastid localized. A microsomal fraction containing recombinant LaPT1 prenylated the isoflavone genistein at the B-ring 3' position to produce isowighteone. The enzyme is also active with 2'-hydroxygenistein but has no activity with other flavonoid substrates. The apparent K(m) of recombinant LaPT1 for the dimethylallyl diphosphate prenyl donor is in a similar range to that of other flavonoid prenyltransferases, but the apparent catalytic efficiency with genistein is considerably higher. Removal of the transit peptide increased the apparent overall activity but also increased the K(m). Medicago truncatula hairy roots expressing LaPT1 accumulated isowighteone, a compound that is not naturally produced in this species, indicating a strategy for metabolic engineering of novel antimicrobial compounds in legumes.     

Fermentation of Insoluble Cellulose by Continuous Cultures of Ruminococcus albus

The hydrolysis and fermentation of insoluble cellulose (Avicel) by continuous cultures of Ruminococcus albus 7 was studied. An anaerobic continuous culture apparatus was designed which permitted gas collection, continuous feeding, and wasting at different retention times. The operation of the apparatus was controlled by a personal computer. Cellulose destruction ranged from ca. 30 to 70% for hydraulic retention times of 0.5 to 2.0 days. Carbon recovery in products was 92 to 97%, and the oxidation-reduction ratios ranged from 0.91 to 1.15. The total product yield (biomass not included) per gram of cellulose (expressed as glucose) was 0.83 g g⁻¹, and the ethanol yield was 0.41 g g⁻¹. The product yield was constant, indicating that product formation was growth linked.     

A Cytokinin Complex in the Developing Fruits of Lupinus albus

The apices of Lupinus albus L. were analysed for cytokinin activity at three stages of development. Little cytokinin activity could be detected in the apices at the time of flowering. However, a considerable amount of activity was detected as the fruits developed. Separate analyses of seed and pod material indicated that there was a high level of cytokinin in both these parts of the fruit. After fractionation of the peaks of activity obtained from paper chromatograms on Sephadex LH-20, four peaks of cytokinin activity were recorded. Two of these co-eluted with zeatin and zeatin riboside. A third peak at an elution volume of 360–440 ml could be hydrolysed with β-glucosidase to give activity at elution volumes corresponding to those of zeatin and zeatin riboside. This strongly suggested that both glucosylated zeatin and glucosylated zeatin riboside were present in the developing fruits of L. albus. The fourth peak at an elution volume of 160–280 ml did not disappear upon hydrolysis with β-glucosidase, and it is possible that it represented a nucleotide cytokinin.     

Changes in lipoxygenase activity during seedling development of Lupinus albus

A lipoxygenase preparation was obtained from Lupinus albus seeds and was shown to differ from previously characterized lipoxygenase. This study describes changes in lipoxygenase activity during seedling development of Lupinus albus. The enzyme activity shows a decrease from 0–6 h postgermination (about 15%), is roughly constant or even rises slightly from 6–30 h and then shows a large increase between 30 and 48 h (about 50%). Enzymatically active proteins from 48 h-old seedlings were isolated and the increase of enzyme activity was mainly due to the presence of two components with maximum activity at pH 6 and pH 8.5, respectively. When arachidonic acid was used as substrate, the two enzymatic activities produce 15 HPETE. The increase in lipoxygenase activity during seedling development was inhibited by cycloheximide. Cordycepin appears to have no direct effect on lipoxygenase synthesis in vivo at the studied doses.     

Effect of drought and rewatering on the metabolism of Lupinus albus organs

Alterations in the metabolism of Lupinus albus organs that result from and subsequently follow a period of severe water deficit (WD) are described. By means of 13C-nuclear magnetic resonance (NMR), changes in the major metabolites were monitored in several plant organs (leaflets and petiole, roots, stem stele and cortex). During the stress, most of the leaves were lost and the stem functioned as a storage repository of sugars (glucose and sucrose) and amino acids (asparagine and proline). Upon rewatering, lupin plants rapidly re-established the relative water content (RWC) and produced new leaves. However, at the metabolic level, the events seem to be more complex, since proline (a stress related metabolite) disappeared rapidly while sugars and asparagine reached the initial pattern more slowly, particularly in the stem.     

Fungitoxic dihydrofuranoisoflavones and related compounds in white lupin,Lupinus albus

Chromatographic investigation of a methanolic extract of white lupin roots has revealed the presence of six new dihydrofuranoisoflavones (lupinisoflavones A-F). Three monoprenylated (3,3-dimethylallyl-substituted) isoflavones (wighteone, luteone and licoisoflavone A), two diprenylated isoflavones [6,3′-di(3,3-dimethylallyl)genistein (lupalbigenin) and 6,3′-di(3,3-dimethylallyl)-2′-hydroxygenistein (2′-hydroxylupalbigenin)] and two pyranoisoflavones (parvisoflavone B and licoisoflavone B) have also been isolated from the same source. In addition to genistein, leaf extracts of L. italbus contain 3′-O-methylorobol which is presumed to be the precursor of lupisoflavone [5,7,4′-trihydroxy-3′-methoxy-6-(3,3-dimethylallyl)isoflavone]. Probable biogenetic relationships between the prenylated, and dihydrofurano-and pyrano-substituted isoflavones in roots and leaves of L. albus are briefly discussed.     

Exocellular (glyco)proteins of Lupinus albus plants and suspension-cultured cells

Plant species from genus Lupinus are among the oldest known legumes, and various aspects of their biology are considerably different from those commonly observed within Leguminosae. To study this issue in more detail, a suspension culture of Lupinus albus cells was developed, and the glycosylation patterns of exocellular proteins analysed. N-linked oligosaccharide side-chains were detected with two lectins: concanavalin A (ConA) and wheat germ agglutinin (WGA) used with respective anti-lectin antibodies, while O-linked arabinosylated side-chains of (hydroxy)proline-rich glycoproteins were identified with anti-(42 kDa French bean chitin-binding protein) antibodies. The obtained data were compared with analogous ones for exocellular (glyco)proteins from suspension-cultured Phaseolus vulgaris cells and from various tissues of L. albus plants. Major species-specific differences between exocellular (glyco)proteins from lupin and bean cells were identified. Similarly, developmentally regulated glycosylation changes following transition from organised plant tissue to dedifferentiated suspension-cultured lupin cells were detected and analysed.