Jan van Brederode

Identification, properties, and genetic control of UDP-glucose: Cyanidin-3-rhamnosyl-(1→6)-glucoside-5-O-glucosyltransferase isolated from petals of the red campion (Silene dioica)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 5-hydroxyl group of cyanidin-3-rhamnosyl-(1→6)-glucoside has been demonstrated in petal extracts of Silene dioica plants. This glucosyltransferase activity was not detectable in green parts of these plants. The enzyme activity is controlled by a single dominant gene M; no glucosyltransferase activity could be demonstrated in petals of m/m plants. The enzyme was purified eightyfold by PVP and Sephadex G50 chromatography. The glucosyltransferase had a pH optimum of 7.4, had a molecular weight of about 55,000, was stimulated by divalent metal ions, and had a “true Km” value of 0.5×10−3m for UDP-glucose and 3.6×10−3m for cyanidin-3-rhamnosylglucoside. Pelargonidin-3-rhamnosylglucoside also could serve as acceptor. The enzyme did not catalyze the glucosylation of the 5-hydroxyl group of cyanidin-3-glucoside, although in petals of M/- n/n mutants cyanidin-3,5-diglucoside is present. ADP-glucose could not serve as a glucosyl donor.

Identification, properties and genetic control of hydroxycinnamoyl-coenzyme A: Anthocyanidin. 3-rhamnosyl (1 → 6) glucoside, 4‴-hydroxycinnamoyl transfersae isolated from petals of Silene dioica

Summary An enzyme catalyzing the transfer of the p -coumaroyl or caffeoyl moiety of respectively p-coumaroyl-CoA and caffeoyl-CoA to the 4-hydroxyl group of the rhamnosyl moiety of anthocyanidin 3-rhamrnosyl(1 → 6)glucosides and 3-rhamosyl(1 → 6)glucoside-5-glycosides has been demonstrated in petal extracts of Silene dioica plants. This acyltransferase activity is governed by gene Ac ; in petals of aclac plants this activity is 15-times lower than in petals of Ac / Ac plants. The enzyme purified fiftyfold by Dowex 1 × 2 and Sephadex G-150 chromatography, exhibits a p H optimum of 7.6-7.8, has a molecular weight of 56,000 daltons, is not stimulated by divalent metal ions, has a “true Km” value of 3.5μM, for caffeoyl-CoA, of 0.29 mM for cyanidin 3-rhamnosyl(1 → 6)glucoside. p -Coumaroyl-CoA can also serve as substrate donor (“true Kin” 15 pill). For the acceptor pelargonidin 3-rhamnosyl(1 → 0)glucosi(le a “true Kin” of 0.03 mM has been obtained.

Identification, Properties and Genetic Control of p-CoumaroylCoenzyme A, 3-Hydroxylase Isolated from Petals of Silene dioica

Summary An enzyme catalyzing the hydroxylation of the 3-position of p -coumaroyl-Coenzyme A has been demonstrated in petal extracts of Silene dioica plants. For optimal activity NADPH, FAD, and molecular oxygen are necessary. The hydroxlating activity is governed by gene P ; in pink petals of p/p plants this activity was absent. In vivo gene P controls both the hydroxylation pattern of the anthocyanin B-ring and that of the acyl group. The enzyme can use p -coumaric acid as non-substrate effector. In this case hydrogen peroxide is formed and p -coumaric acid remains unchanged during the course of the reaction. Although the enzyme isolated from petals of p/p plants was not able to hydroxylate p -coumaroyl- CoA, it could still use p -coumaric acid as non-substrate effector.

Identification and properties of UDP-glucose: cyanidin-3-O-glucosyltransferase isolated from petals of the red campion (Silene dioica).

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been demonstrated in petal extracts of Silene dioica mutants with cyanidin-3-O-glucoside in the petals. This transferase activity was also present in young rosette leaves and calyces of these plants. The highest glucosyltransferase activity was found in petals of opening flowers of young plants. The enzyme was purified ninetyfold by PVP and Sephadex chromatography. The glucosyltransferase had a pH optimum of 7.5, had a “true Km value” of 4.1×10−4m for UDP-glucose and 0.4×10−4m for cyanidin chloride, and was not stimulated by divalent metal ions. Both p-chloromercuribenzoate and HgCl2 inhibited the enzyme activity. Pelargonidin chloride and delphinidin chloride at reduced rates also served as substrates. The enzyme did not catalyze the glucosylation of the 3-hydroxyl group of flavonols or the 5-hydroxyl group of anthocyanins. ADP-glucose could not serve as a glucosyl donor. The results of Sephadex G150 chromatography suggest that the glucosyltransferase can exist as dimer of about 125,000 daltons and as active monomers of 60,000 daltons. The genetic control of the glucosyltransferase activity is discussed.

The formation of UDP‐L‐rhamnose from UDP‐D‐glucose by an enzyme preparation of red campion (Silene Dioica (L) clairv) leaves

In microorganisms at least three enzymes are involved in the conversion of TDP-D-glucose to TDP-Lrhamnose [I ,2]. The formation of TDP-4-keto-6deoxy-D-glucose, the first demonstrable intermediate, is catalyzed by TDP-D-glucose-4,6-hydrolyase (EC 4.2.1.43). This enzyme [3,4], which consists of two subunits firmly bound by one molecuie NAD’, initially attacks TDP-D-glucose at C-4 to yield TDP-4keto-D-glucose and enzyme-NADH. This TDP-4-ketoD-glucose then rearranges by &elimination of water between C-5 and C-6 to form an unsaturated glucoseen, which serves as hydrogen acceptor for the enzyme-NADH, and leads to the intermediate TDP-4keto-6-deoxy-D-glucose. Possibly via the ene-diol form, TDP+keto-6-deoxyD-glucose; 3,5-epimerase catalyzes the epimerizations at C-3 and C-S. The epimerizations are followed by a stereospecitic reduction by NADPH: TDP-6-deoxyL-lyxo4hexulose; 4-reductase. The 3,5-epimerase and 4-reductase are sometimes considered to be one single enzyme: TDP-L-rhamnose synthetase (see also fig.1) [S]. In plants the conversion of TDP-D-glucose to TDP-L-rhamnose proceeds with a very low efficiency ]6,7]. With UDP-D-glucose, however, the forlnation of UDP-L-rhamnose takes place at an appreciable higher rate [7]. It is not known whether in plants the synthesis proceeds according to the same reaction mechanism as in microorganisms. We here want to describe the conversion of UDP-Dglucose to UDP-L-rhamnose in Silene dioica and

The 2″-O-glucosylation of vitexin and isovitexin in petals of Silene alba is catalysed by two different enzymes

Two separate genes, Fg and Vg, which govern the presence of isovitexin 2″-O-glucoside and vitexin 2″-O-glucoside respectively in the petals of Silene alba control different glucosyltransferases. In Vg/Vg,fg/fg plants no isovitexin 2″-O-glucosyltransferase was present and in vg/vg,Fg/Fg plants no vitexin 2″-O-glucosyltransferase activity could be detected. The Fg-controlled UDP-glucose: isovitexin 2″-O-glucosyltransferase has a pH optimum of8.5, while the Vg-controlled vitexin 2″- O-glucosyltransferase has a pH optimum of7.5. Both glucosyltransferases are stimulated by the divalent cations Ca2+, Co2+, Mn2+ and Mg2+. For isovitexin 2″-O-glucosylation, however, much higher concentrations are needed than for vitexin 2″-O-glucosylation.For UDP-glucose a ‘true Km’ value of0.3 mM with the Fg-controlled and of 0.2 mM with the Vg-controlled enzyme was found. For isovitexin and vitexin these values are respectively 0.09 and 0.01 mM.

A pattern analysis of the geographical distribution of flavone-glycosylating genes in Silene pratensis

Abstract The three flavone glycosylating genes in European Silene pratensis shows a distinct geographical variation. Three genetically different races can be distinguished on the basis of gene frequencies. Pattern recognition techniques and cluster analysis have been used on the data set of frequencies of the flavone-glycosylating genes in 285 European populations of S. pratensis . The combined results of these analyses confirm the previously recognized races and reveal a steep cline between races 7 and 2(+3) and a wide wide transition zone between races 2 and 3.

Variation in Crassulacean acid metabolism within the genus Sedum : carbon isotope composition and water status dependent phosphoenolpyruvate carboxylase activity

Summary Variation in Crassulacean acid metabolism (CAM) was examined in a large group of Sedum species, using two criteria: 13 C/ 12 C ratio and water status dependent specific activity of the CAM key enzyme phosphoenolpyruvate carboxylase (PEP-C). A large amount of variation in CAM was found within the genus Sedum . In three European species C3-like photosynthesis was found, as indicated by water status independent, low specific PEP-C activity and δ 13 C values of around -30‰. Three Mexican species showed water status independent high specific PEP-C activity and δ 13 C values of -15 to -22‰, indicating constitutive CAM. In the majority of European species the specific PEP-C activity was water status dependent; δ 13 C values ranged from -25 to -31‰. Specific PEP-C activity and carbon isotope ratios are significantly correlated, species with high specific activities showing less negative δ 13 C values. The Michaelis constant for PEP of the PEP-C enzymes of these Sedum species was determined. The observed values differed from 120-800 µmo1. The observed variation in CAM was compared with taxonomic relations of the examined species, leaf thickness, Km(PEP) and life cycle length.

Isozyme Variation in Silene pratensis: a Response to Different Environments

The isozymes of nine enzyme systems were screened and the frequencies of the flavone glycosylating genes were determined in an outdoor experiment with 70 populations of European S. pratensis and an indoor experiment with 30 populations of the same species. Cluster analysis (using Wards cluster criterion) were performed on all data sets. In the outdoor experiment, cluster analysis of the flavonoid data gave the same pattern that we obtained in an ealier survey of a larger set of populations and showed clearly that there are eight chemical races in European S. pratensis. No comparable geographic distributiion could be found in the isozyme data set, although the dendrogram showed two very clearly separated groups. The two groups represented the two years in which the populations were grown. This result indicates that isozyme variation in European S. pratensis is largely determined by environmental factors. Observations on changes in isozyme patterns during ontogeny and on differences between indoor and outdoor grown plants of the same F2 crosses confirm this. Differences in isozyme patterns can be caused by very small differencs in environment as is shown by the results of the indoor experiment, in which a slight gradient in environmental conditions was present in the greenhouses. The cluster analysis of the isozyme data from the indoor experiment revealed three distinct groups of populations that could be related to location within the greenhouses. As in the outdoor experiment, the dendrogram for the flavonoids gave the same geographic pattern as found with the earlier survey.

Biosynthesis and genetic control of isovitexin 2″-O-arabinoside in petals of Silene dioica

Abstract In petals of Silene dioica plants, an enzyme has been demonstrated which catalyses the transfer of the arabinose moiety of UDP-arabinose to the hydroxyl group on the 2″-position of the carbon-carbon bound glucose of isovitexin. The presence of this arabinosyltransferase activity is controlled by the dominant allele gl A . Maximal activity takes place at pH 7.2–7.5; the reaction is stimulated by the divalent metal ions Mg and Mn. For optimal solubilization of the enzyme, Triton X-100 is necessary. Substrate specificity and kinetic behaviour have been investigated.