Oxidation products of acylated anthocyanins under acidic and neutral conditions

Acylated anthocyanidin-3,5-diglucosides are oxidized with H₂O₂ under acidic conditions to acylated ortho-benzoyloxyphenylacetic acid esters. When the same reaction is carried out under neutral conditions, the reaction product is the 3-O-acyl-glucosyl-5-O-glucosyl-7-hydroxy coumarin.     

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.     

Enzymatic control of anthocyanin expression in the flowers of pea (Pisum sativum) mutants

Using enzymological and immunological methods we have investigated the relationship between chalcone synthase and the A locus, a major gene involved in the control of anthocyanin expression in pea (Pisum sativum L.) flowers. Pea plants containing the dominant allele A usually synthesize anthocyanins in the petal tissue, whereas plants homozygous for the a allele do not produce anthocyanins. We sought to determine whether or not the A locus also controlled the presence or absence of chalcone synthase, the first enzyme of the flavonoid pathway in the flowers of three genetic lines (A, purple-violet flowers; A,am, white flowers with sometimes pink edges; and a, white flowers). Chalcone synthase was found to be present in all three genetic lines by enzyme activity measurement, indirect enzyme-linked immunosorbent assay (ELISA), and Western blotting. Spectroscopic investigations showed that only the genetic lines A and A,am contained anthocyanins and flavonol glycosides, respectively, in the flowers; line a accumulated p-coumaric acid or its derivatives. These data suggest that the A locus in Pisum is not the structural gene for chalcone synthase and it does not appear to regulate the expression of this enzyme.This work was supported by a grant from the Cornell University Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation and a consortium of industries.     

Light induced changes in anthocyanin concentration,activity of phenylalanine ammonia-lyase and flavanone synthase and some of their properties in Brassica oleracea

Seedlings of red cabbage, Brassica oleracea cv Red Danish, germinated in the dark, rapidly produced anthocyanins upon illumination. The anthocyanin production increased up to six days of illumination time. The activity of phenylalanine ammonia-lyase increased rapidly in illuminated seedlings to a maximum at 8 hr and declined thereafter to dark levels. During this period the activity of flavanone synthase, the first enzyme responsible for the establishment of C₁₅ flavonoid skeleton, paralleled that of the anthocyanin concentration. The crude flavanone synthase has a pH optimum at around 8, a molecular weight of ca 120 000, and is able to utilize only p-coumaryl-CoA as co-substrate for the production of flavonoids.     

Vacuolar contents of fruit subepidermal cells from vitis species

Enzymic treatment of mature, anthocyanin-containing grape berry sub-epidermal tissues from DeChaunac grapes released intact protoplasts. Filtration of the protoplast suspension through glass wool under mild suction resulted in the release of vacuoles. The total and individual contents of anthocyanins, flavonol glycosides, hydroxycinnamic acid esters, sugars, organic acids, and cations were determined in both tissue and vacuole preparations. A method for pH determination in intact vacuoles is reported. Based on the qualitative and quantitative anthocyanin composition, the average pH of the vacuoles was determined to be 2.7 (sd +/- 0.17). The data suggest that anthocyanins are present in fruit subepidermal tissues in a noncomplexed form.     

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.     

Subcellular localization of enzymes of anthocyanin biosynthesis in protoplasts

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.     

Subcellular localization of flavonoid synthesizing enzymes in Pisum,Phaseolus, Brassica and Spinacia cultivars

Functionally-intact chloroplasts were obtained from 11-day-old pea (Pisum sativum cv Midfreezer) seedlings. Enzyme-distribution studies with ribulose bisphosphate carboxylase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase showed that ca 2.1% of the total tissue chloroplasts were present in the chloroplast preparation. The rate of intactness of chloroplast preparations was 34–82%. SAM:caffeic acid methyltransferase, flavanone synthase, UDPG:flavonoid-3-O-glucosyltransferase and SAM:quercetin methyltransferase activities were measured in the homogenate, supernatant and chloroplast lysate fractions. Significant activities of the above four enzymes could only be detected in the homogenate and supernatant fractions. Similar experiments with 11-day-old seedlings of green beans (Phaseolus vulgaris cv Early Gallatin), red cabbage (Brassica oleracea cv Red Danish) and 6-week-old plants of spinach (Spinacia oleracea cv Bloomsdale) showed a similar distribution of the flavonoid synthesizing enzymes. We conclude that under the reported conditions chloroplasts are not involved in flavonoid biosynthesis.     

Chalcone Synthase Localization in Early Stages of Plant Development. I. Immunohistochemical Use of Plasmolysis for Localizing the Enzyme in Epidermal Cell Cytoplasm of Illuminated Buckwheat Hypocotyls

Immunohistochemical methods combined with progressive plasmolysis were used to localize chalcone synthase (CHS), an important enzyme for plant metabolism of aromatics in hypocotyls of illuminated buckwheat (Fagopyrum esculentum M) seedlings. Illumination of etiolated seedlings with white light results in anthocyanin synthesis in the epidermal layer of the hypocotyl. Anthocyanin-containing epidermal peels, after fixation for 30 min in 4% paraformaldehyde, 2.5% glutaraldehyde, 0.1% caffeine, were treated with a specific rabbit anti-buckwheat CHS antibody and a 20 nm goat anti-rabbit IgG gold conjugate. CHS is specifically shown in epidermal cells as pink to dark red deposits. Progressive plasmolysis combined with our immunohistochemical method showed that CHS was located exclusively in the cytoplasm of the epidermal cells of buckwheat hypocotyls except for the guard cells, which contained no detectable CHS.