Cloning, expression and characterisation of two tyrosinase cDNAs from Agaricus bisporus.

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.

The uncoupling efficiency and affinity of flavonoids for vesicles.

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.

Preheating effects on the textural strength of canned green beans. 1. Cell wall chemistry.

Variable preheating conditions allowed the modification of the firmness of two green bean cultivars after processing. The aim of this study was to elucidate the biochemical basis of this phenomenon and to relate pectin differences to different inherent firmness of two cultivars. The preheating temperature, which resulted in the highest retention of firmness after sterilization, corresponded with the optimal temperature for pectin methylesterase activity. After this preheating treatment, there was an overall reduction of the degree of methylation of the cell wall pectin. In addition, the yields of the buffer and chelator soluble fractions, as well as their average molecular mass, were higher after sterilization. Firmness differences between the two cultivars seemed to be related to the degree of methylation, the degree of acetylation, and the total amount of pectins. Preheating of green beans affects texture after sterilization most likely by demethylation of pectin by pectin methylesterase thereby (i) decreasing the beta-eliminative degradation of pectin and (ii) increasing the capacity of pectin to form Ca(2+)-mediated complexes.

Isolation and partial characterization of thermostable isoperoxidases from potato (Solanum tuberosum L.) tuber sprouts.

Peroxidases (POD; EC 1.11.1.7) can cross-link cell wall polymers and may have an impact on the final textural quality of potato tubers. Because heat treatments are important during processing, the thermal properties of isoPODs from soluble and ionically and covalently bound fractions were studied from both potato tubers and sprouts. For both tissues, the ionically bound fraction was the most thermostable; approximately 20% of POD activity remained after a heat treatment of 10 min at 90 degrees C (for sprouts). The temperature profile of the ionically bound sprout fraction appeared to be nonlinear and suggested the presence of a very thermostable POD, which still showed activity after a heat treatment at 100 degrees C. Visualization by using isoelectric focusing confirmed the occurrence of a thermostable isoPOD with an IEP of 9.5, which displayed regeneration of activity after heat inactivation. This cationic POD was further purified by chromatography techniques, and by SDS-PAGE its molecular mass was estimated at 38 kDa.

Changes in cell wall polysaccharides of green bean pods during development.

The changes in cell wall polysaccharides and selected cell wall-modifying 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, beta-galactosidase, and alpha-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.

Characterization of pectinases and pectin methylesterase cDNAs in pods of green beans (Phaseolus vulgaris L.).

Tomato fruit maturation is accompanied by a depolymerization of cell wall pectins which is due to the action of endopolygalacturonase (endoPG) preceded by pectin methylesterase (PE) activity. To investigate the role of endoPG and PE in determining the structure of green bean (Phaseolus vulgaris L.) pectins, these pectinases were studied during pod development. Early developmental stages displayed low endoPG or exoPG activities while PE activities were measurable during all stages of pod and seed development. These results do not favour a possible synergistic action of PE and PG. For seeds, the relatively high PE activities concurred with relatively low levels of pectin methyl esterification. At a molecular level, one partial chromosomal clone of 210 bp (PE1V), two partial PE cDNA clones of 660 bp (PE2V and PE3V) from cv. verona and one full-length PE cDNA clone of 1990 bp (PE3M), from cv. Masai were isolated. The identity of the CDNA clones was confirmed by expression in Escherichia coli and immunodetection with antibodies directed towards a tomato fruit PE. Transcripts corresponding with the genomic clone PE1V were not detected but both PE2 and PE3 cDNAs corresponded with mRNAs 1.8 kb in length. In contrast to PE2, PE3 gene expression levels varied significantly in pods from different cultivars suggesting an involvement in determining pod morphology.

Activation of flavonoid biosynthesis in roots of Vicia sativa subsp. nigra plants by inoculation with Rhizobium leguminosarum biovar viciae.

Infective (nodulating) Rhizobium leguminosarum biovar viciae (R.l. viciae) bacteria release Nod factors which stimulate the release of nodulation gene-inducing flavanones and chalcones from roots of the host plant Vicia sativa subsp. nigra (K. Recourt et al., Plant Mol Biol 16: 841-852; H.P. Spaink et al., Nature 354: 125-130). The hypothesis that this release results from increased synthesis of flavonoids was tested by studying the effect of inoculation of V. sativa with infective and uninfective R.l. viciae bacteria on (i) activity of L-phenylalanine ammonia-lyase, (ii) level of chalcone synthase mRNA, and (iii) activity of (eriodictyol) methyltransferase in roots. Consistent with the hypothesis, each of these parameters was found to increase 1.5 to 2-fold upon inoculation with infective R.l. viciae bacteria relative to the situation for uninoculated roots and for roots inoculated with uninfective rhizobia.

Major flavonoids in uninoculated and inoculated roots of Vicia sativa subsp. nigra are four conjugates of the nodulation gene-inhibitor kaempferol.

Inoculation of Vicia sativa subsp. nigra (V. sativa) roots with Rhizobium leguminosarum biovar. viciae (R.l. viciae) bacteria substantially increases the ability of V. sativa to induce rhizobial nodulation (nod) genes. This increase is caused by the additional release of flavanones and chalcones which all induce the nod genes of R.l. viciae (K. Recourt et al., Plant Mol Biol 16: 841-852). In this paper, we describe the analyses of the flavonoids present in roots of V. sativa. Independent of inoculation with R.l. viciae, these roots contain four 3-O-glycosides of the flavonol kaempferol. These flavonoids appeared not capable of inducing the nod genes of R.l. viciae but instead are moderately active in inhibiting the activated state of those nod genes. Roots of 7-day-old V. sativa seedlings did not show any kaempferol-glycosidase activity consistent with the observation that kaempferol is not released upon inoculation with R.l. viciae. It is therefore most likely that inoculation with infective (nodulating) R.l. viciae bacteria results in de novo flavonoid biosynthesis and not in liberation of flavonoids from a pre-existing pool.

Inoculation of Vicia sativa subsp. nigra roots with Rhizobium leguminosarum biovar viciae results in release of nod gene activating flavanones and chalcones.

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.

Regulation and manipulation of flavonoid gene expression in anthers of petunia: the molecular basis of the Po mutation.

Molecular mechanisms governing development of the male reproductive organs of flowers, the anthers, are largely unknown. In this article, we report on the investigation of the molecular basis of a mutation involving the expression of a gene encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase (CHI) in anthers of petunia. In petunia, the gene Po regulates the expression of CHI in anthers: PoPo petunia lines contain CHI enzyme activity in petals and anthers, whereas popo lines contain the CHI enzyme only in petals but not in anthers. As a result of the Po mutation, the substrate of CHI accumulates and therefore the pollen of a popo line are yellow or greenish. The genome of petunia contains two chi genes, chiA and chiB. In a restriction fragment length polymorphism analysis, a 100% linkage was observed between Po and chiA. This result suggested that Po is identical to chiA and that Po is not a regulatory gene of chiA. Introduction of a chiA gene isolated from a PoPo line into a popo line resulted in a complementation of the mutation that was directly visible because the pollen color shifted from yellow to white. This proved that chiA and Po are identical. Because chiA encodes a functional CHI enzyme in flower petals of a popo line, we propose that the Po mutation is a mutation in the regulatory region of chiA abolishing chiA promoter activity in anthers but not in corollas. This change in anther color is a fine illustration of how floral pigmentation can be manipulated in a predictable way and suggests the use of CHI as a visible marker.

A biovar-specific signal of Rhizobium leguminosarum bv. viciae induces increased nodulation gene-inducing activity in root exudate of Vicia sativa subsp. nigra.

Flavonoids in root exudate of leguminous plants activate the transcription of Rhizobium genes involved in the formation of root nodules (nod genes). We report that inoculation with the homologous symbiont R. leguminosarum bv. viciae results in an increased nod gene-inducing activity (Ini) in root exudate of V. sativa subsp. nigra, whereas inoculation with heterologous Rhizobium strains results in exudates with nod gene-inducing activity comparable to that of uninfected plants. Ini can be demonstrated by using either of the isogenic indicator strains containing an inducible nod promoter fused to the Escherichia coli lacZ reporter gene and the regulatory nodD gene of R. leguminosarum bv. viciae, R. leguminosarum bv. trifolii, or R. meliloti. The presence of genes nodDABCEL of R. leguminosarum bv. viciae appeared to be essential for induction of Ini. Mutation of the genes nodI and nodJ causes a delay of Ini, whereas gene nodF appears to be required for both the timely appearance and the maximum level of Ini activity. The nodE gene is responsible for the biovar specificity of induction of Ini by Rhizobium spp. Ini is caused by a soluble heat-stable factor of rhizobial origin. This Rhizobium-produced Ini factor has an apparent molecular weight between 1,000 and 10,000 and does not originate from flavonoid precursors.

Accumulation of a nod gene inducer, the flavonoid naringenin, in the cytoplasmic membrane of Rhizobium leguminosarum biovar viciae is caused by the pH-dependent hydrophobicity of naringenin.

Most Sym plasmid-localized nodulation genes of Rhizobium leguminosarum bv. viciae are only expressed upon activation of the NodD protein by plant flavonoids, e.g., naringenin (S. A. J. Zaat, C. A. Wijffelman, H. P. Spaink, A. A. N. van Brussel, and B. J. J. Lugtenberg, J. Bacteriol, 169:198-204, 1987). As part of a study on the mechanism of NodD protein activation, the mechanism of uptake and the intracellular fate of [3H]naringenin were studied. Naringenin was accumulated by Rhizobium cells without apparent metabolic conversion to an 80-fold-higher concentration in a process which did not require any of the other Sym plasmid-localized nod genes. Naringenin accumulation was nonsaturable, highly reversible, and not inhibited by the presence of other flavonoids or the metabolic inhibitors potassium cyanide, sodium azide, 2,4-dinitrophenol, and carbonyl cyanide m-chlorophenylhydrazone. These data indicate an accumulation mechanism without high affinity sites which does not use cellular energy. In vitro, naringenin has high affinity for the cytoplasmic membrane. This binding was pH dependent, very high at pH 5.7 and not present anymore at pH 9.7. A similar pH dependency was found for the affinity of naringenin for the olive oil fraction of a biphasic olive oil-water system. pH-dependent changes in the UV spectrum indicate ionization of naringenin at high pH to a negatively charged form. Since it has recently been shown that the nodD gene product is located in the cytoplasmic membrane (H. R. M. Schlaman, H. P. Spaink, R. J. H. Okker, and B. J. J. Lugtenberg, J. Bacteriol., in press), our data are consistent with a model in which the un-ionized form of naringenin accumulates in the cytoplasmic membrane and activates, in a metabolically unaltered form, the NodD protein.

Cloning and characterization of a gene encoding an outer membrane protein required for siderophore-mediated uptake of Fe3+ in Pseudomonas putida WCS358.

In iron-limited environments plant-growth-stimulating Pseudomonas putida WCS358 produces a yellow-green fluorescent siderophore called pseudobactin 358. Ferric pseudobactin 358 is efficiently taken up by cells of WCS358 but not by cells of another rhizophere-colonizing strain, Pseudomonas fluorescens WCS374. A gene bank containing partial Sau3A DNA fragments from WCS358 was constructed in a derivative of the broad-host-range cosmid pLAFR1. By mobilization of this gene bank to strain WCS374 a cosmid clone, pMR, which made WCS374 competent for the utilization of pseudobactin 358 was identified. By subcloning of the 29.4-kilobase (kb) insert of pMR the essential genetic information was localized on a BglII fragment of 5.3 kb. Tn5 mutagenesis limited the responsible gene to a region of approximately 2.5 kb within this fragment. Since the gene encodes an outer membrane protein with a predicted molecular mass of 90,000 daltons, it probably functions as the receptor for ferric pseudobactin 358. The gene is flanked by pseudobactin 358 biosynthesis genes on both sides and is on a separate transcriptional unit. WCS374 cells carrying pMR derivatives with Tn5 insertions in the putative receptor gene did not produce the 90,000-dalton protein anymore and were unable to take up Fe3+ via pseudobactin 358. In WCS358 cells as well as in WCS374 cells the gene is expressed only under iron-limited conditions.

Siderophore-mediated uptake of Fe3+ by the plant growth-stimulating Pseudomonas putida strain WCS358 and by other rhizosphere microorganisms.

Under iron-limited conditions, Pseudomonas putida WCS358 produces a siderophore, pseudobactin 358, which is essential for the plant growth-stimulating ability of this strain. Cells of strain WCS358, provided that they have been grown under Fe3+ limitation, take up 55Fe3+ from the 55Fe3+-labeled pseudobactin 358 complex with Km and Vmax values of 0.23 microM and 0.14 nmol/mg of cell dry weight per min, respectively. Uptake experiments with cells treated with various metabolic inhibitors showed that this Fe3+ uptake process was dependent on the proton motive force. Furthermore, strain WCS358 was shown to be able to take up Fe3+ complexed to the siderophore of another plant-beneficial P. fluorescens strain, WCS374. The tested pathogenic rhizobacteria and rhizofungi were neither able to grow on Fe3+-deficient medium in the presence of pseudobactin 358 nor able to take up 55Fe3+ from 55Fe3+-pseudobactin 358. The same applies for three cyanide-producing Pseudomonas strains which are supposed to be representatives of the minor pathogens. These results indicate that the extraordinary ability of strain WCS358 to compete efficiently for Fe3+ is based on the fact that the pathogenic and deleterious rhizosphere microorganisms, in contrast to strain WCS358 itself, are not able to take up Fe3+ from Fe3+-pseudobactin 358 complexes.