P. de Vlaming

Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida.

Petunia hybrida mutants, homozygous recessive for one of the genes An1, An2, An6, or An9 do not show anthocyanin synthesis in in vitro complementation experiments per se (see also Kho et al. 1977). Extracts of flowers of these mutants all provoke anthocyanin synthesis in isolated petals of an an3an3 mutant. Mutants homozygous recessive for one of the genes An1, An2, An6, or An9 and homozygous recessive for F1 accumulate dihydroflavonols in comparable amounts. The synthesis of dihydromyricetin is blocked in an1an1 mutants, which indicates a regulating effect of the gene An1 on the gene Hfl. Similar mutants, but dominant for F1, accumulate flavonols (kaempferol and quercetin) instead of dihydroflavonols. Myricetin is accumulated in minor amounts and not at all in an1an1 mutant. Furthermore, the synthesis of this flavonol is not controlled by the gene F1. The synthesis of cyanidin (derivatives) is greatly reduced when flavonols are synthesized (F1 dominant). In mutants dominant for Ht1 and Hf1 and thus able to synthesize cyanidin (derivatives) and delphinidin (derivatives), predominantly delphinidin (derivatives) are synthesized. The results indicate that kaempferol (derivatives), quercetin (derivatives), and delphinidin (derivatives) are the main endproducts of flavonoid biosynthesis in Petunia hybrida.

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.

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.

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.

Genetic and biochemical studies on the conversion of flavanones to dihydroflavonols in flowers of Petunia hybrida

SummaryChemogenetic investigations and precursor experiments on flowers of Petunia hybrida suggest that recessive alleles of the gene An3 block the biosynthetic pathway of flavonols and anthocyanins between the flavanone and dihydroflavonol step. In confirmation of this hypothesis, activity of the enzyme flavanone 3-hydroxylase, which catalyses the conversion of flavanones to dihydroflavonols, was readily demonstrated in enzyme preparations from flowers of lines with the dominant allele An3, whereas no or very low activity could be found in extracts from lines with recessive alleles (an3an3). A second genetic factor is described which clearly reduces the amount of flavonols in the flowers but not the amount of anthocyanins. Crossing experiments revealed that this factor represents a third allele of the An3 gene. It is referred to as an3-1. As expected, a residual flavanone 3-hydroxylase activity of about 10% could be found in enzyme extracts from plants with the an3-1 allele. The decreased level of dihydroflavonol formed under this condition is obviously still sufficient for anthocyanin formation but not for flavonol synthesis.Similar to flavanone 3-hydroxylases from other plants, the enzyme of Petunia is a soluble enzyme and belongs according to its cofactor requirements to the 2-oxoglutarate-dependent dioxygenases. The residual flavanone 3-hydroxylase activity found in plants with the an3-1 allele is identical to the activity extracted from An3-genotypes with regard to cofactors, substrate specificity and most of the inhibitors. The difference observed in the respective pH-optima and the genetic data suggest that the mutation providing the an3-1 phenotype is localized in the structural gene for flavanone 3-hydroxylase.

Genetic control of anthocyanin-O-methyltransferase activity in flowers of Petunia hybrida

SummaryThe relation between four methylation genes (Mt1, Mt2, Mf1 and Mf2) in flowers of Petunia hybrida and anthocyanin-methyltransferase activity was investigated in vitro. All genes controlled methyltransferase activity. This activity was measured with cyanidinnd petunidin-derivatives as substrates. A cross provided evidence that the Mf-genes regulate methyltransferases which are distinct from those controlled by the Mtgenes. Different effects of the two Mf-genes in vivo are shown. The results suggest that the four methylationgenes control four different methyltransferases.

A gene for flower colour fading in Petunia hybrida

SummaryFull coloured Petunia hybrida flowers lose their colour and become completely white if the gene Fa is dominant. This gene is only expressed in mutants with the pH gene Ph4 homozygous recessive. It is shown by genetical experiments that the fading is restricted to the 3-rutinosido(p-coumaroyl)-5glucoside glycosylation pattern of anthocyanins. The 3-glucosides and 3-rutinosides show a weak fading only.