Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.     

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 (K_m 0.22 mM), the enzyme glucosylated the 3-(p-coumaroyl)-rutinoside derivatives of delphinidin and petunidin (K_m 3 M), 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.Abbreviations HPLC high performance liquid chromatography - 3GT 3-O-glucosyltransferase - 5GT 5-O-glucosyltransferase - 3RGac 3-(p-coumaroyl)-rutinoside - 3RGac5G 3-(p-coumaroyl)-rutinoside-5-glucoside - UDPGlc uridine 5-diphosphoglucose     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

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

Summary Chemogenetic 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.     

A gene controlling rate of anthocyanin synthesis and mutation frequency of the gene An1 in Petunia hybrida

Summary The difference in colour intensity between flowers of sporogenic revertants of the white flowering lines W17 and W28 is caused by an incompletely dominant gene Inl. This gene is not linked to the anthocyanin gene Anl. In the dominant state Inl causes a 50% decrease in colour intensity of selfcoloured red flowers.Chromatographic analysis of anthocyanins of plants homozygous recessive or dominant for Inl showed that the same anthocyanins are produced in both genotypes (cyanidin-3-glucoside and cyanidin-3-diglucoside). Anthocyanin synthesis starts at the same stage of development of the flower in both genotypes. When the bud reaches a length of approximately 45 mm, however, anthocyanin synthesis in the Inl Inl line slows down.No influence of the gene Inl on the concentration of dihydroquercetin-7-glucoside in buds and flowers could be observed, which indicates that the influence of Inl on flower colour development is restricted to the last part of the biosynthesis of anthocyanins, i.e. the conversion of dihydroflavonols into anthocyanins.In addition to Inl having a decreasing effect on flower colour intensity, evidence is produced that the gene Inl also influences the reversion frequency of unstable alleles of the gene Anl.     

Ethylene sensitivity: The role of short-chain saturated fatty acids in pollination-induced senescence of Petunia hybrida flowers

Normal and pollination-induced senescence of Petunia hybrida L cv. Pink Cascade flowers is accompanied by an increase in the sensitivity of the corolla to ethylene as indicated by an acceleration in the rate of corolla bluing after exposure to exogenous ethylene. Pollination resulted in the production of short-chain saturated fatty acids ranging in chain length from C₆ to C₁₀. Following pollination, these acids are synthesized in the stylar tissue via the acetate pathway within the first 12 hours. The fatty acids are transported rapidly to the corolla where they induce an increase in ethylene sensitivity. In unpollinated flowers, these acids are produced in the corolla during the early stages of senescence. Although the levels of these fatty acids decrease rapidly during the final stages of senescence, a significant increase in ethylene sensitivity could be detected prior to the decrease. It appears that the increase in ethylene sensitivity caused by the synthesis of short-chain saturated fatty acids occurs concurrently, but independent from ethylene synthesis.     

Genetic analysis of instability in Petunia hybrida

Summary In 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.     

An effective chemical mutagenesis procedure for Petunia hybrida cell suspension cultures

Summary By using mercury(II)-chloride (HgCl₂) and Dl-6-fluorotryptophan (6FT) as positive selection conditions we were able to show that N-methyl-N-nitro-N-nitrosoguanidine (NG) is an effective mutagen for Petunia hybrida suspension cells.A number of the 205 calli resistant to HgCl₂ and 17 calli resistant to 6FT were isolated. The highest mutation frequency was 1.0 x 10^–5 and 2.0 x 10^–6 for HgCl₂ and 6FT, respectively. A preliminary characterization of the mutants is presented.A significant increase in the number of drug-resistant calli was only obtained at NG-concentrations (5–40 g/ml) that had no observable effect on the survival of the mutagenized cultures.     

Genetic analysis of instability in Petunia hybrida

Summary The effect of environmental factors on the reversion rates of several unstable alleles in Petunia hybrida was investigated. It is demonstrated that the reversion frequency of three unstable alleles, viz. an allele of gene An1 and of gene An11, both involved in anthocyanin synthesis, and of gene Yg3 for leaf colour, is drastically reduced when the temperature is raised from 18 °C to 25 °C. For two of the alleles it was established that this temperature effect is reversible. Changing the light period or light intensity did not have an effect on the reversion rate of the unstable allele of gene An11 at 18 °C or at 25 °C. The results found are in contrast with those obtained in earlier experiments, in which a rise in temperature resulted in an increase in the reversion rate of another unstable allele of gene An1.     

On the origin of anthocyanin methyltransferase isozymes of Petunia hybrida and their role in regulation of anthocyanin methylation

Summary Four genes controlling anthocyanin methylation in flowers of Petunia hybrida have been described. Three of them, Mt2, Mf1 and Mf2, caused a dosage effect on anthocyanin methyltransferase activity and degree of methylation of anthocyanins. Antiserum raised against partially purified Mf2-enzyme precipitated three of the four anthocyanin methyltransferases. In two subspecies of one of the ancestral species of P. hybrida: Petunia integrifolia, different anthocyanin methyltransferases were found as determined by immunoprecipitation. The methyltransferase isozymes showed no differences in subcellular or tissue location, and had no physiologically important difference in time course of activity during bud development. The methylation-system in Petunia is discussed with regard to anthocyanin methylation in other plant species.     

Identification of a S-ribonuclease-binding protein in Petunia hybrida

To investigate protein-protein interactions in gametophytic self-incompatibility, we used a yeast two-hybrid assay to identify proteins that could interact with the S-ribonuclease protein. These assays identified a pollen-expressed protein, which we have named PhSBP1, that appears to bind with a high degree of specificity to the Petunia hybrida S-ribonuclease. Although PhSBP1 activates reporter gene expression only when expressed in tandem with a S-RNAse bait protein, binding is not allele-specific. Sequence analysis demonstrated that PhSBP1 contained a C-terminal cysteine-rich region that includes a RING-HC domain. Because many RING-finger domain proteins appear to function as E3 ubiquitin ligases, our results suggest that ubiquitination and protein degradation may play a role in regulating self-incompatibility interactions. Together, these results suggest that PhSBP1 may be a candidate for the recently proposed general inhibitor (RI) of self-incompatibility ribonucleases.     

Isolation and characterization of anthocyanin variants originating from the unstable system an2-1 in Petunia hybrida (Hort.)

Summary Forty stable an2-n alleles, derived from the unstable system an2-1, have been tested for anthocyanin synthesis. All of them proved to be different from both the An2 and an2 natural alleles. Only two were distinct from the others which according to Duncan's multiple range test formed a group of overlapping populations. Amongst the variants isolated there was a large majority of light-coloured types. Regulation-like effects of the an2-n alleles on the subsequent genes of the anthocyanin pathway have been observed. A hypothesis concerning the nature of the genetic events occurring at the An2 locus is discussed.     

Self-incompatibility alleles control a low molecular weight,basic protein in pistils of Petunia hybrida

Summary Proteins extracted from the pistils of several clones of Petunia hybrida carrying differing pairs of S alleles were examined by gel electrophoresis. The major protein of pistils, a basic glycoprotein of relatively low molecular weight, showed properties which varied in a simple manner with the S genotype. For each S allele we were able to assign a specific molecular weight (ranging from 27,000 to 33,000) and isoelectric point (in the range 8.3 to 8.7) for this putative S protein. Pistils homozygous at the S locus showed only one major protein on two-dimensional gel electrophoresis, while pistils from plants heterozygous at the S locus showed two. No evidence was obtained for the presence of this putative S protein in pollen extracts.     

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Summary Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0–28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.     

Petunia hybrida S-proteins: ribonuclease activity and the role of their glycan side chains in self-incompatibility

Summary Self-incompatibility in flowering plants is controlled by the S-gene, encoding stylar S (allele-specific) glycoproteins. In addition to three previously characterized Petunia hybrida S-proteins, we identified by N-terminal sequence analysis another stylar S-protein, co-segregating with the S _b-allele. Purified S-proteins reveal biological activity, as is demonstrated for two of them by the allele-specific inhibition of pollen tube growth in vitro. Moreover, the four isolated S-proteins are ribonucleases (S-RNases). Specific activities vary from 30 (S₁) to 1000 (S₂) units per min per mg protein. We attempted to investigate the functionality of the carbohydrate portion of the S-RNases. Deglycosylation studies with the enzyme peptide-N-glycosidase F (PNGase F) reveals differences in the number of N-linked glycan chains present on the four S-RNases. Variability in the extent of glycosylation accounts for most of the molecular weight differences observed among these proteins. By amino acid sequencing, the positions of two of the three N-glycosylation sites on the S₂-RNase could be located near the N-terminus. Enzymic removal of the glycan side chains has no effect on the RNase activity of native S-RNases. This suggests another role of the glycan moiety in the self-incompatibility mechanism.     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

S allele discrimination in styles of Petunia hybrida bearing stylar-conditioned pseudo-self-compatibility

Summary A cross between a 0% pseudo-self-compatible (PSC) plant (S_3.3) and a 100% PSC plant (S_1.1) yielded an F₁ population which, when selfed, produced a high mean seed set which was not significantly different than that produced when the F₁ was backcross pollinated by the 100% PSC parent. Backcross pollinating the F₁ with the 0% PSC parent yielded no seed. No S_3.3 plants were recovered in the F₂ populations, indicating that pollen tubes containing the S₃ allele were inhibited during pollen tube growth of the selfed F₁ plants. Apparently stylar-conditioned PSC does not remove all discriminatory power from these petunia styles. Crossing the F₁ (S_1.3) with an self-incompatible (SI) plant (S_2.2) produced plants which were used for computation of a standard linkage test. An approximate map distance of 28 units was found between the S specificity locus and the major gene(s) which influenced its expression. Other generalized PSC modifying genes apparantly were not linked with the S locus.Scientific Journal Series Paper Number 10,606 of the Minnesota Agricultural Experiment Station     

Regulation of gene action in Petunia hybrida: Unstable alleles of a gene for flower colour

Summary In a progeny of a selfed individual of the dark red-flowered cultivar Roter Vogel some white-flowered plants appeared as the result of a mutation of the genetic factor Anl involved in anthocyanin synthesis. The white flowers of these plants had red spots owing to back-mutations in the dermal cells of the young corolla.Owing to a striking unstability of the new allele of Anl, a number of mutants originated which differ mutually in the frequency of reversion, which expressed itself in the very substantial differences in the spot density of the limb of the corolla. Between a mean number of less than one spot per cm² of the limb and a mean number of over 10.000 spots/cm², a series of transitions was found.The reversions did not remain restricted to the young epidermis but also occurred in sporogenous tissues. This resulted in the appearance of selfcoloured red descendants of plants with red-spotted white flowers. There is a positive correlation between the spot density of the parent plants and the percentage of plants with completely red corollas.The red spots on the corolla usually have the same colour as the wild type (Roter Vogel), but occasionally mutants occur with paler spots, the colour varying from a very pale pink to a red nearly as deep as in the wild type. The selfcoloured descendants of such mutants also show this colour variation from pale pink to red.On the grounds of these observations a theory was formulated which postulates that the Anl locus consists of a structural gene responsible for an enzyme active during anthocyanin synthesis and a regulatory element built up from intermediate repetitive DNA. This regulatory element in turn is built up of two components, one of which, the mutator, decides the activation of the structural gene while the other, the expressor, modifies the rate of activation. The mutations must be considered representative of larger or smaller deletions within one or both of these components. Reversions are the result of the restoration of the deletions by means of an amplification of the repetitive DNA in dividing cells of the developing flower buds.