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The hydroxylation of p-coumaric acid by an enzyme from leaves of spinach beet (Beta vulgaris L.).

ABSTRACT: 1. An enzyme from the leaves of spinach beet (Beta vulgaris L.) that catalyses the hydroxylation of p-coumaric acid to caffeic acid in the presence of ascorbate has been purified about 1000-fold on a protein basis. 2. It is activated by high concentrations of ammonium sulphate and sodium chloride. 3. The preparation shows both hydroxylase and catechol oxidase activities, in a constant ratio throughout the purification procedure; they are similarly activated by salts. 4. Ascorbate acts as a reductant in quantities equivalent to the caffeic acid produced by hydroxylation. 5. Ascorbate can be replaced by tetrahydrofolic acid, NADH, NADPH or 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not by caffeic acid. Among these, the pteridine is the most effective, but the reaction is not inhibited by aminopterin. In experiments with saturating concentrations of NADH and the pteridine, these reductants compete in the reaction and are equivalent on a molar basis. 6. No cofactor has been separated from the enzyme by prolonged dialysis. 7. The relation of the enzyme to other hydroxylases and phenolases is discussed.

SUBMITTER: Vaughan PF

PROVIDER: S-EPMC1184609 | biostudies-other | 1969 Jun

REPOSITORIES: biostudies-other

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The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.).

Project description:1. Under defined conditions, the hydroxylation of p-coumaric acid catalysed by a phenolase from leaves of spinach beet (Beta vulgaris L.) was observed to develop its maximum rate only after a lag period. 2. By decreasing the reaction rate with lower enzyme concentrations or by increasing it with higher concentrations of reductants, the length of the lag period was inversely related to the maximum rate subsequently developed. 3. Low concentrations of caffeic acid or other o-dihydric phenols abolished this lag period. With caffeic acid, the rate of hydroxylation was independent of the reductant employed. 4. Hydroxylation was inhibited by diethyldithiocarbamate, but with low inhibitor concentrations hydroxylation recovered after a lag period. This lag could again be abolished by the addition of high concentrations of caffeic acid or other o-dihydric phenols. 5. Catechol oxidase activity showed no lag period, and did not recover from diethyldithiocarbamate inhibition. 6. The purified enzyme contained 0.17-0.33% copper; preparations with the highest specific activity were found to have the highest copper content. 7. The results are interpreted to suggest that the oxidation of o-dihydric phenols converts the enzymic copper into a species catalytically active in hydroxylation. This may represent the primary function for the catechol oxidase activity of the phenolase complex. The electron donors are concerned mainly, but not entirely, in the reduction of o-quinones produced in this reaction.

| S-EPMC1179322 | biostudies-other

Kinetic studies on the hydroxylation of p-coumaric acid to caffeic acid by spinach-beet phenolase.

Project description:1. A spectrophotometric assay is described that enables the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, to be followed continuously. 2. Initial-velocity and inhibitor studies indicate that the order of substrate addition is oxygen, p-coumaric acid and electron donor, with an irreversible step separating the binding of each substrate. 3. Caffeic acid is most likely to act as electron donor at the active site; other electron donors, such as ascorbic acid, NADH and dimethyltetrahydropteridine, function mainly to recycle cofactor amounts of caffeic acid. 4. A reaction scheme, consistent with these data, is proposed.

| S-EPMC1165638 | biostudies-other

The action of hydrogen peroxide on the hydroxylation of p-coumaric acid by spinach-beet phenolase.

Project description:Treatment of spinach-beet phenolase with H2O2 under aerobic conditions results in a stimulation of the p-coumaric acid hydroxylation it catalyses, but not the caffeic acid oxidation. Spectroscopic evidence suggests that an oxygenated enzyme species is formed under these conditions.

| S-EPMC1172427 | biostudies-other

The expression of catechol oxidase activity during the hydroxylation of p-coumaric acid by spinach-beet phenolase.

Project description:1. The conditions under which oxygen consumption in excess of that required for the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, can be suppressed, have been examined. 2. With dimethyltetrahydropteridine as electron donor, oxygen uptake was exactly equivalent to the caffeic acid produced, provided that p-coumaric acid was in excess, but with excess of reductant, oxygen uptake caused by the further oxidation of caffeic acid was also observed. 3. With equal concentrations of ascorbate and p-coumaric acid, equivalent oxygen uptake and caffeic acid production was found only in the first stages of the reaction, whereas with NADH substituted for ascorbate, oxygen uptake was in excess throughout. 4. When ascorbate was used, the period of the reaction over which this equivalence was found was decreased at high reaction rates and not observed at all with aged enzyme preparations; equivalence was restored by adding bovine serum albumin to these aged preparations. 5. Equivalence between oxygen consumption and caffeic acid production was observed with NADH, if small quantities of dimethyltetrahydropteridine were also added. 6. It is concluded that hydroxylation proceeds without the concomitant production of caffeic acid only if the enzyme is stabilized for hydroxylation by p-coumaric acid and the reductant, and is protected from attack by o-quinones.

| S-EPMC1178761 | biostudies-other

Proteomic Profiling of Sugar Beet (Beta vulgaris) Leaves during Rhizomania Compatible Interactions.

Project description:Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV), severely impacts sugar beet (Beta vulgaris) production throughout the world, and is widely prevalent in most production regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with BNYVV, but as resistance breaking strains become more prevalent, effective disease control strategies will require the application of novel methods based on better understanding of disease susceptibility and symptom development. Herein, proteomic profiling was conducted on susceptible sugar beet, infected with two strains of BNYVV, to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugar beet leaf tissue infected with BNYVV, quantified, and analyzed by mass spectrometry. A total of 203 proteins were confidently identified, with a predominance of proteins associated with photosynthesis and energy, metabolism, and response to stimulus. Many proteins identified in this study are typically associated with systemic acquired resistance and general plant defense responses. These results expand on relatively limited proteomic data available for sugar beet and provide the ground work for additional studies focused on understanding the interaction of BNYVV with sugar beet.

| S-EPMC5302737 | biostudies-literature

Enzymatic Browning in Sugar Beet Leaves (Beta vulgaris L.): Influence of Caffeic Acid Derivatives, Oxidative Coupling, and Coupled Oxidation.

Project description:Sugar beet (Beta vulgaris L.) leaves of 8 month (8m) plants showed more enzymatic browning than those of 3 month (3m). Total phenolic content increased from 4.6 to 9.4 mg/g FW in 3m and 8m, respectively, quantitated by reverse-phase-ultrahigh-performance liquid chromatography-ultraviolet-mass spectrometry (RP-UHPLC-UV-MS). The PPO activity was 6.7 times higher in extracts from 8m than from 3m leaves. Substrate content increased from 0.53 to 2.45 mg/g FW in 3m and 8m, respectively, of which caffeic acid glycosyl esters were most important, increasing 10-fold with age. Caffeic acid glycosides and vitexin derivatives were no substrates. In 3m and 8m, nonsubstrate-to-substrate ratios were 8:1 and 3:1, respectively. A model system showed browning at 3:1 ratio due to formation of products with extensive conjugated systems through oxidative coupling and coupled oxidation. The 8:1 ratio did not turn brown as oxidative coupling occurred without much coupled oxidation. We postulate that differences in nonsubstrate-to-substrate ratio and therewith extent of coupled oxidation explain browning.

| S-EPMC5481818 | biostudies-literature

Characterisation of sugar beet (Beta vulgaris L. ssp. vulgaris) varieties using microsatellite markers.

Project description:BackgroundSugar beet is an obligate outcrossing species. Varieties consist of mixtures of plants from various parental combinations. As the number of informative morphological characteristics is limited, this leads to some problems in variety registration research.ResultsWe have developed 25 new microsatellite markers for sugar beet. A selection of 12 markers with high quality patterns was used to characterise 40 diploid and triploid varieties. For each variety 30 individual plants were genotyped. The markers amplified 3-21 different alleles. Varieties had up to 7 different alleles at one marker locus. All varieties could be distinguished. For the diploid varieties, the expected heterozygosity ranged from 0.458 to 0.744. The average inbreeding coefficient F(is) was 0.282 +/- 0.124, but it varied widely among marker loci, from F(is) = +0.876 (heterozygote deficiency) to F(is) = -0.350 (excess of heterozygotes). The genetic differentiation among diploid varieties was relatively constant among markers (F(st) = 0.232 +/- 0.027). Among triploid varieties the genetic differentiation was much lower (F(st) = 0.100 +/- 0.010). The overall genetic differentiation between diploid and triploid varieties was F(st) = 0.133 across all loci. Part of this differentiation may coincide with the differentiation among breeders' gene pools, which was Fst = 0.063.ConclusionsBased on a combination of scores for individual plants all varieties can be distinguished using the 12 markers developed here. The markers may also be used for mapping and in molecular breeding. In addition, they may be employed in studying gene flow from crop to wild populations.

| S-EPMC2890681 | biostudies-literature

Beta vulgaris ssp. vulgaris chromosome 8 shows significant association with geosmin concentration in table beet.

Project description:Geosmin, a degraded sesquiterpene molecule with earthy and musty odor, imbues table beet with its characteristic aroma. Geosmin is heritable and endogenously produced in table beet; its earthy aroma is sought by some consumers but deters others. Geosmin biosynthesis is catalyzed by a bifunctional geosmin synthase enzyme in diverse bacteria and fungi, but a mechanism for geosmin biosynthesis in plants has not been reported. This work employed association analysis and selective genotyping of a segregating F2:3 mapping population to seek QTL associated with geosmin concentration in table beet. GBS reads were aligned to sugar beet reference genome EL10.2, and association analysis revealed two QTL for geosmin concentration on Beta vulgaris ssp. vulgaris chromosome 8. QTL at EL10.2 positions 28,017,624 and 38,488,687 each show effect size 8.7 μg·kg-1 geosmin and explain 8.5% and 6.4% of total variation in geosmin concentration, respectively. Resolution was low due to large recombination bin size and imperfect alignment between the reference genome and mapping population, but population size and selection proportion were sufficient to detect moderate to large effect QTL. This study, the first molecular genetic mapping experiment in table beet, succeeded in finding QTL for geosmin concentration in table beet, and it provides the basis for fine mapping or candidate gene investigation of functional loci for this distinctive sensory trait.

| S-EPMC8664477 | biostudies-literature

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| S-EPMC6668331 | biostudies-literature

Sugar Beet (Beta vulgaris) Guard Cells Responses to Salinity Stress: A Proteomic Analysis.

Project description:Soil salinity is a major environmental constraint affecting crop growth and threatening global food security. Plants adapt to salinity by optimizing the performance of stomata. Stomata are formed by two guard cells (GCs) that are morphologically and functionally distinct from the other leaf cells. These microscopic sphincters inserted into the wax-covered epidermis of the shoot balance CO2 intake for photosynthetic carbon gain and concomitant water loss. In order to better understand the molecular mechanisms underlying stomatal function under saline conditions, we used proteomics approach to study isolated GCs from the salt-tolerant sugar beet species. Of the 2088 proteins identified in sugar beet GCs, 82 were differentially regulated by salt treatment. According to bioinformatics analysis (GO enrichment analysis and protein classification), these proteins were involved in lipid metabolism, cell wall modification, ATP biosynthesis, and signaling. Among the significant differentially abundant proteins, several proteins classified as "stress proteins" were upregulated, including non-specific lipid transfer protein, chaperone proteins, heat shock proteins, inorganic pyrophosphatase 2, responsible for energized vacuole membrane for ion transportation. Moreover, several antioxidant enzymes (peroxide, superoxidase dismutase) were highly upregulated. Furthermore, cell wall proteins detected in GCs provided some evidence that GC walls were more flexible in response to salt stress. Proteins such as L-ascorbate oxidase that were constitutively high under both control and high salinity conditions may contribute to the ability of sugar beet GCs to adapt to salinity by mitigating salinity-induced oxidative stress.

| S-EPMC7212754 | biostudies-literature

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