Using primers designed on the basis of sequence homologies in the copper-binding domains for a number of plant and fungal tyrosinases, two tyrosinase encoding cDNAs were cloned from an Agaricus bisporus U1 cDNA-library. The sequences AbPPO1 and AbPPO2 were, respectively, 1.9 and 1.8 kb in size and encoded proteins of approximately 64 kDa. The cDNAs represent different loci. Both AbPPO1 and AbPPO2 occur as single copies on the genomes of the U1 parental strains H39 and H97. The genomic size of AbPPO1 and AbPPO2 is minimally 2.3 and 2.2 kb, respectively. Alignment and phylogenetic analysis of 35 tyrosinase and polyphenol oxidase sequences of animal, plant, fungal, and bacterial origin indicated conserved copper-binding domains, and stronger conservation within genera than between them. The translation products of AbPPO1 and AbPPO2 possess putative N-glycosylation and phosphorylation sites and are recognised by antibodies directed against a 43-kDa tyrosinase. The observations are consistent with previously proposed maturation and activation models for plant and fungal tyrosinases.

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394-5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using 1H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.

Cell wall material (CWM) was isolated from noncooked, 5 min cooked, and 15 min cooked potato tissue and accompanying cooking media from the cultivars Irene and Nicola. A mass balance of the material in the different fractions obtained during isolation was made. Chemical compositions of the CWM and cell size distributions of the potato tissues were analyzed. The mealy cooking cv. Irene had more CWM per unit cell surface area than the nonmealy cooking cv. Nicola. These results confirmed observations of the potato cell walls made by transmission electron microscopy. The molar composition of the CWM was comparable for both cultivars. During cooking, for both cultivars relatively more unbranched than branched pectic polysaccharides were solubilized. However, the type of pectin solubilized after 15 min of cooking was different for the two cultivars. This pectin was relatively more branched, more methylated, and more acetylated for cv. Irene than for cv. Nicola. Keywords: Potato; Solanum tuberosum L.; cooking; texture; cell wall; middle lamella; transmission electron microscopy (TEM)

The changes in cell wall polysaccharides and selected cell wallmodifying enzymes were studied during the development of green bean (Phaseolus vulgaris L.) pods. An overall increase of cell wall material on a dry-weight basis was observed during pod development. Major changes were detected in the pectic polymers. Young, exponentially growing cell walls contained large amounts of neutral, sugar-rich pectic polymers (rhamnogalacturonan), which were water insoluble and relatively tightly connected to the cell wall. During elongation, more galactose-rich pectic polymers were deposited into the cell wall. In addition, the level of branched rhamnogalacturonan remained constant, while the level of linear homogalacturonan steadily increased. During maturation of the pods, galactose-rich pectic polymers were degraded, while the accumulation of soluble homogalacturonan continued. During senescence there was an increase in the amount of ionically complexed pectins, mainly at the expense of freely soluble pectins. The most abundant of the enzymes tested for was pectin methylesterase. Peroxidase, ␤-galactosidase, and ␣-arabinosidase were also detected in appreciable amounts. Polygalacturonase was detected only in very small amounts throughout development. The relationship between endogenous enzyme levels and the properties of cell wall polymers is discussed with respect to cell wall synthesis and degradation.The texture of processed vegetables and fruits is in part determined by the properties of the cell wall and the middle lamella (Stolle-Smits et al., 1997). The cell wall is not a static structure, it is dynamic in nature. Its composition and structure change continuously during plant development. Plant cell walls consist of cellulose microfibrils coated by xyloglucans and embedded in a complex matrix of pectic polysaccharides (Talbot and Ray, 1992;Carpita and Gibeaut, 1993). Pectic substances are abundant in fruit and vegetable cell walls and are considered to be important in determining the texture of processed vegetables. Cell wall pectin consists of two regions, a linear homogalacturonan (HGA) and a branched rhamnogalacturonan. Neutral side chains, mainly consisting of arabinosyl and/or galactosyl residues, are attached to the rhamnogalacturonan backbone in variable amounts. In addition, xylosyl units can be linked to the GalUA backbone, as was reported for branched apple pectins (Schols et al., 1995).RGII is a very typical branched pectin that occurs only in minor amounts and is suggested to function as a signal molecule (Darvill et al., 1978). The carboxyl and hydroxyl groups of the GalUA backbone can be substituted with methyl and acetyl esters, respectively. Pectin is believed be connected to other cell wall components or other pectins by ionic and covalent cross-links (Brett and Waldron, 1990). The exact nature of these cross-links is still unknown, but cross-linking esters are thought to be involved. The aim of this study was to analyze the modifications of cell wall composition in green bean (Phaseolus vulgaris ...

The relative hydrophobicity and interaction of flavonoids with artificial membranes using vesicles was studied. At the same degree of hydroxylation, flavones were slightly more hydrophobic than flavanones. Flavonoids possess a hydrophobic character and are weak acids. For this reason, their uncoupling efficiency of the membrane potential was studied using cytochrome c oxidase vesicles. With emphasis on naringenin, it was shown that flavonoids affect both the transmembrane potential difference (V) and the transmembrane pH difference (V). Flavones were slightly more effective in uncoupling the membrane potential than flavanones; the 7OH group seems to play an important role. Hydroxylation of the exocyclic phenyl group decreased the uncoupling efficiency for all flavonoids studied. The flavonol quercitin exhibited hardly any uncoupling activity. Glycosylation abolished all uncoupling activity. The affinity of flavonoids for vesicle membranes was also studied using the fluorescence quenching of the membrane probe diphenylhexatriene. Flavonols exhibited a substantially higher affinity for liposomes than flavanones. This difference in affinity is assumed to be caused by the far more planar configuration of the flavonols in comparison with the tilted configuration of flavanones. Due to this planar configuration, it seems reasonable to assume that flavonols could more easily intercalate into the organised structures of the phospholipids within the vesicle membranes than flavanones. It is concluded that, in vivo, hardly any uncoupling activity of flavonoids can be anticipated. However, the quercitin plasma concentration in vivo can be such that, based on the affinity study, part of this flavonol could be associated with biological membranes to function there as, for example, an antioxidant.