A. W. Schram

Regulation of flavonoid gene expression in Petunia hybrida: Description and partial characterization of a conditional mutant in chalcone synthase gene expression

SummaryWhite flowers of the Petunia hybrida line W43 accumulate glucosides of 4-coumaric acid and caffeic acid and are able to synthesize anthocyanins from exogeneously supplied naringenin, suggesting that W43 is blocked in a biosynthetic step preceding the formation of naringenin. The cultivar Red Star contains a similar mutation as W43; the genetic background of this cultivar, however, allows the production of considerable amounts of anthocyanins in certain areas of the flower. When grown at reduced light, flowers of Red Star are uniformly coloured, whereas under an enhanced light regime the flowers exhibit alternating white and coloured areas.In white sectors of flowers with a colour pattern virtually no chalcone synthase (CHS) enzyme activity could be demonstrated. The enzymes chalcone isomerase (CHI), UDPG: flavonoid-3-0-glucosyltransferase (3GT) and SAM: anthocyanin methyltransferase (OMT) are present, although at a more or less reduced level. From Western blotting and in vitro translation experiments we infer that the absence of CHS enzyme activity in the white sectors of the cultivar Red Star is due to the absence of translationally active CHS mRNA. The potential use of the mutants for genetic engineering in plants is emphasized.

The identity of α-galactosidase B from human liver

Abstract 1. 1. The identity of α-galactosidase B (α- d -galactoside galactohydrolase, EC 3.2.1.22), the minor α-galactosidase isoenzyme present in normal human tissues and the component responsible for the residual α-galactosidase activity in patients with Fabrys disease, was investigated. For this investigation, α-galactosidase B was purified from normal human liver. 2. 2. Purified α-galactosidase B contains α-glucosidase, α-xylosidase, N-acetyl-αgalactosaminidase and α-galactosidase activity, as measured with p-nitrophenyl glycosides as substrates. 3. 3. Incubation of purified α-galactosidase B with an antiserum against the same preparation resulted in a reduction of both α-galactosidase and N-acetyl-αgalactosaminidase activities. The extent of the reduction was dependent on the amount of antiserum added, an identical titration curve being obtained with both activities. On the other hand, the α-glucosidase activity was not affected by incubation with the antiserum and α-xylosidase was inactivated by incubation either with antiserum or with normal serum. 4. 4. Incubation of purified α-galactosidase B at 50°C for 4 h led to about 34% inactivation of both α-galactosidase and N-acetyl-α-galactosaminidase. 5. 5. The Km of purified α-galactosidase B for p-nitrophenyl-α-galactoside (about 20 mM) is higher than that for p-nitrophenyl-N-acetyl-α-galactosaminide (about 1 mM). The maximal velocity with the latter substrate is about 2.3-fold higher than that with the former. 6. 6. When purified α-galactosidase B is incubated with p-nitrophenyl-α-galactoside and p-nitrophenyl-N-acetyl-α-galactosaminide together, the rate of p-nitrophenol formation is considerably less than the sum of the rates observed when the substrates are added singly. 7. 7. It is concluded that the α-galactosidase and N-acetyl-α-galactosaminidase activities in purified α-galactosidase B are due to the same protein containing a single catalytic site.

Genes affecting flower colour and pH of flower limb homogenates in Petunia hybrida

SummaryIn Petunia hybrida four complementary genes are present, each having, if homozygous recessive a blueing effect on the flower colour. These genes have no qualitative or quantitative effect on anthocyanins and flavonols. In mutants homozygous recessive for one (or more) of the Ph genes the pH of aqueous flower limb homogenates is increased. It is assumed that the Ph genes in Petunia are involved in maintaining the pH in the vacuole fluid in the flower.

Properties and genetic control of anthocyanin 5-O-glucosyltransferase in flowers of Petunia hybrida

An anthocyanin 5-O-glucosyltransferase from flowers of Petunia hybrida was purified about 30-fold. Using uridine 5′-diphosphoglucose as glucose donor (Km 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (Km 3 μM), isolated from pollen of Petunia. Delphinidin 3-rutinoside, cyanidin 3-rutinoside and delphinidin 3-glucoside did not serve as substrates. The glucosylation of petunidin 3-(p-coumaroyl)-rutinoside showed a pH-activity optimum at pH 8.3 and was neither stimulated by Mg2+ or Ca2+, nor inhibited by ethylenediaminetetraacetic acid. After separating the 5-O-glucosyltransferase from the anthocyanidin 3-O-glucosyltransferase by means of chromatofocusing, it was shown that both enzymes exhibit a high degree of positional specificity. The 5-O-glucosyltransferase activity was correlated with the gene An1, but not with the gene Gf.

Inheritance and Biochemistry of Pigments

Since the introduction of Petunia axillaris (Lam.) B.S.P. and P. integrifolia (Hook.) Sch. et Th. in Europe between 1823 and 1835, crossings have been made between the two species (Bailey 1896, Ferguson and Ottley 1932). The first results of these experiments, with regard to flower color, are pictured in 1837 by both Harrison and Hooker. It is impossible to explain those results in terms of genes, although the pictures allow us to attribute some presently known alleles to them. Also, the experiments of Naudin (1858, 1865) and Hoffmann (1869), done before the rediscovery of Mendelian laws, have only historical value.

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

SummaryIn flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3′-position. Similar to the flavonoid 3′-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3′-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3′-position in Petunia. Thus, the introduction of the 3′-hydroxyl group is clearly achieved by hydroxylation of C15-intermediates, and the concomitant occurrence of the 3′,4′-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.

Properties and genetic control of four methyltransferases involved in methylation of anthocyanins in flowers of Petunia hybrida

Four S-adenosyl-l-methionine:anthocyanin-3′,5′-O-methyltransferases in flowers of Petunia hybrida were separated using the chromatofocusing technique. Each methyltransferase is controlled by one of the methylation genes Mt1, Mt2, Mf1 or Mf2. Molecular weight, pH-activity optimum, isoelectric point, several kinetic properties and the behaviour in the presence of Mg2+, ethylenediaminetetraacetic acid and S-adenosyl-l-homocysteine of each of the four enzymes were determined. The methylation in vitro of delphinidin 3-(p-coumaroyl)-rutinosido-5-glucoside reflected the accumulation patterns of methylated anthocyanins in vivo and established the regulatory role of methyltransferases in vivo.

Genetic analysis of instability in Petunia hybrida : 2. Unstable mutations at different loci as the result of transpositions of the genetic element inserted at the An1 locus.

SummaryIn crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.

The genetic control of the enzyme UDP-glucose: 3-0-flavonoïd-glucosyltransferase in flowers of Petunia hybrida

SummaryFour genes controlling the conversion of dihydroflavonols into anthocyanins have been investigated for their effect on UDP-Glucose: 3-0-flavonoïd glucosyltransferase activity, one of the enzymes involved in this conversion. An1 and An2 control the bulk of UFGT activity; a homozygous recessive for one of these genes shows an activity of 5–20% of the wildtype value.In a homozygous double recessive some 5% activity is still found while in mutants homozygous recessive for An6 or An9, UFGT activity is lower. In F2 progenies segregating for An6 or An9, however, no difference in UFGT activity was found between homozygous recessive and dominant plants.Mutants blocked in a biosynthesis step preceding the formation of dihydroflavonols show normal UFGT activity levels, indicating that no anthocyanidins are needed for UFGT induction. In addition to delphinidin, myricetin was used as a substrate. The results obtained indicate the probability that both substrates can be glucosylated by the same UFGT enzyme.

Methylation of anthocyanins by cell-free extracts of flower buds of Petunia hybrida

Abstract An O -methyltransferase activity which catalyses the methylation of anthocyanins was extracted from flowerbuds of Petunia hybrida . The methyltransferase uses S -adenosyl- l -methionine as methyl donor. Only anthocyanidin 3( p -coumaroyl)rutinosido-5-glucoside was methylated. No methylating activity towards anthocyanidins, anthocyanidin 3-glucosides, anthocyanidin 3-rutinosides, caffeic acid or p -coumaric acid could be detected.