Geza Hrazdina

Metabolic pathways as enzyme complexes: Evidence for the synthesis of phenylpropanoids and flavonoids on membrane associated enzyme complexes

In earlier studies [G. Hrazdina, G. J. Wagner, and H. W. Siegelman (1978) Phytochemistry 17, 53-56; G. J. Wagner and G. Hrazdina (1984) Plant Physiol. 74, 901-906], evidence was obtained suggesting that the endoplasmic reticulum was a site for phenylpropanoid and flavonoid metabolism in petal tissue, and that (a) multienzyme complex(es) might be involved in this metabolism. Now, the possible role of membrane-bound multienzyme complexes in phenylpropanoid and flavonoid metabolism in three tissues has been investigated by (1) correlating enzyme induction kinetics and rates, (2) examining the molecular weight of putative complexes, (3) channeling of substrates, (4) determining the susceptibility of bound activities to trypsin digestion, and (5) investigating the structurally linked latency of bound activities. Results suggest that at least a part--and possibly the entire pathway--from phenylalanine to flavonoids is membrane (endoplasmic reticulum) associated, and that this metabolism is facilitated by a multienzyme complex. Phenylalanine ammonia lyase, the first enzyme of the biosynthetic sequence, and a flavonoid glucosyltransferase, the last, appear to be located in the lumen of the membranes. Cinnamate 4-hydroxylase is membrane embedded, while other enzyme activities appear to be weakly associated with the cytoplasmic face of endoplasmic reticulum membranes.

Stress responses in alfalfa (Medicago sativa L.) 11. Molecular cloning and expression of alfalfa isoflavone reductase, a key enzyme of isoflavonoid phytoalexin biosynthesis

The major phytoalexin in alfalfa is the isoflavonoid (−)-medicarpin (or 6aR, 11aR)-medicarpin. Isoflavone reductase (IFR), the penultimate enzyme in medicarpin biosynthesis, is responsible for introducing one of two chiral centers in (−)-medicarpin. We have isolated a 1.18 kb alfalfa cDNA (pIFRalf1) which, when expressed in Escherichia coli, converts 2′-hydroxyformononetin stereospecifically to (3R)-vestitone, as would be predicted for IFR from alfalfa. The calculated molecular weight of the polypeptide (35400) derived from the 954 bp open reading frame compares favorably to estimated Mrs determined for IFR proteins purified from other legumes. The transcript (1.4 kb) is highly induced in elicited alfalfa cell cultures. The kinetics of induction are consistent with the appearance of IFR activity, the accumulation of medicarpin, and the observed induction of other enzymes in the pathway. Low levels of IFR transcripts were found in healthy plant parts (roots and nodules) which accumulate low levels of a medicarpin glucoside. IFR appears to be encoded by a single gene in alfalfa. The cloning of IFR opens up the possibility of genetic manipulation of phytoalexin biosynthesis in alfalfa by altering isoflavonoid stereochemistry.

Substrate specificity of flavanone synthase from cell suspension cultures of parsley and structure of release products in vitro

Abstract The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p -Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4′-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys. 169 , 84–90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p -hydroxybenzalacetone. The apparent K m values for malonyl-CoA and p -coumaroyl-CoA in the reaction leading to naringenin, and for p -coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 μ m , respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p -methoxycinnamoyl-CoA, isoferuloyl-CoA, or p -hydroxybenzoyl-CoA. None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo . The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p -coumaroyl-CoA.

Structural aspects of anthocyanin-flavonoid complex formation and its role in plant color

Abstract The complex formation of flavonoids with anthocyanins, resulting in increase in both absorbance and in a bathochromic shift of the visible absorption maximum of the latter, is based mainly on hydrogen bond formation between the carbonyl group of the anthocyanin anhydrobase and aromatic hydroxyl groups of the complex-forming flavonoids. The larger the number of hydroxyl groups in the flavonoid molecule, the stronger the complex formation. The presence of a 3-hydroxyl group in the flavonoid molecule has little effect on the complex-forming ability. The nature of the sugar substituent of the complex-forming flavonoid compound has no influence on the reaction. The 5-hydroxyl group of flavonoids is strongly bound by intramolecular hydrogen bond to the 4-carbonyl and does not participate in the complex formation. The most important hydroxyl group in the flavonoid molecule is the one in the 7-position. Unsaturation at C2C3 in the heterocyclic ring is an important factor for complex formation. Aromatic hydroxyl groups in the flavonoid system alone cannot account for all the complex-forming ability, suggesting additional involvement by electrostatic forces and configurational or steric effects.

Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts

Abstract Flavanone synthase, chalcone-flavanone isomerase and UDP-glucose; anthocyanidin-3- O -glucosyltransferase activities of protoplasts and subcellular fractions of protoplasts of Hippeastrum and Tulipa were investigated. Subcellular fractions studied were intact vacuoles, cytosol and particulate components of protoplasts less the vacuole. The cytosol fraction had the highest activity of the three enzymes studied. Results similar to those found for Hippeastrum were obtained with fractions from leaves and petals of Tulipa . The increase in flavanone synthase activity in the cytosol fraction from petals of Hippeastrum during development paralleled the increase in anthocyanin content of the petals.

Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

Abstract Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures, was not observed with the H. gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation.

Endopolygalacturonase in Apples (Malus domestica) and Its Expression during Fruit Ripening

The activity of polygalacturonase (PG) has been detected in ripe McIntosh apples (Malus domestica Borkh. cv McIntosh) both by enzyme activity measurement and immunoblotting using an anti-tomato-PG antibody preparation. PG activity increased during fruit ripening and remained steady, or decreased slightly, after 5 months of controlled atmospheric storage. The enzyme had a relative molecular weight of 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 56,000 to 61,000 when determined by gel filtration. Viscosity and reducing end group measurements with a commercial pectin preparation showed that the enzyme is endo acting. In RNA and DNA blot hybridization experiments, a full-length tomato PG cDNA hybridized with the apple RNA and DNA, showing the identity of genes encoding the activity of the enzyme in tomato and apple.

Anthocyanin composition of Brassica oleracea cv. Red Danish

Abstract Eight anthocyanins were isolated from illuminated red cabbage seedlings. They were identified as: cyanidin-3-sophoroside-5-glucoside, cyanidin-3-malonyl-sophoroside-5-glucoside, cyanidin-3-p-coumaryl-sophoroside-5-glucoside, cyanidin-3-(di-p-coumaryl)sophoroside-5-glucoside, cyanidin-3-ferulyl-sophoroside-5-glucoside, cyanidin-3-(diferulyl)sophoroside-5-glucoside, cyanidin-3-sinapylsophoroside-5-glucoside, and cyanidin-3-(disinapyl)-sophoroside-5-glucoside.

Quercetin: A mutagen, not a carcinogen, in fischer rats

Purified quercetin, as well as diets containing quercetin at 0.1% and 0.2%, are mutagens to Salmonella typhimurium TA100. This mutagenicity is enhanced with the S9 metabolic activation system. The urine of Fischer rats fed the 0.2% quercetin diet also is mutagenic with the S9 activation system, but the feces of these animals exhibited enhanced mutagenicity only without activation. This may indicate different quercetin metabolites in urine and feces. Rats fed these diets for 64 wk showed no consistent tissue lesions, carcinogenicity, or reproductive changes. Male rats fed 0.2% quercetin showed lowered blood serum glutamic oxaloacetic transaminase and urea nitrogen levels, but these values do not reflect pathological changes.

High-pressure liquid chromatographic separation of 3-glucosides, 3,5-diglucosides, 3-(6-O-p-coumaryl)glucosides and 3-(6-O-p-coumarylglucoside)-5-glucosides of anthocyanidins

Abstract A high-pressure liquid chromatomagraphic method has been developed for separation of anthocyanins from fruits and their products on a μBondapak c 18 analytical column. Separation of 3- and 3,5-diglucosides f anthocyanidins can be achieved with aqueous acetic acid. That of the p -coumaryl-3 and 3,5-diglucosides requires an aqueous methanolic acetic acid solution. For the separation of complex mixtures of anthocyanins containing members of all four above mentioned pigment groups, a programmed non-linear-gradient elution between aqueous acetic acid and aqueous methanolic acetic solution is required. This technique enables a relatively fast separation and identification of twenty anthocyains in one run without prior treatment of the plant extract, or derivatization of the piments.