The multiple phenylpropene synthases in both Clarkia breweri and Petunia hybrida represent two distinct protein lineages

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.     

Metabolic Regulation of Floral Scent in Petunia axillaris Lines: Biosynthetic Relationship between Dihydroconiferyl Acetate and iso-Eugenol

Aromatic scent-related compounds in flowers were comprehensively analyzed by high-performance liquid chromatography (HPLC) based on their absorption spectra to understand regulation of metabolism leading to floral scent diversity in Petunia axillaris lines. An unknown compound occurring at similar levels to scent compounds in some plant lines was identified to be dihydroconiferyl acetate. Based on the structure, dihydroconiferyl acetate is likely to be a biosynthetically closely related compound to aromatic scent compounds, especially iso-eugenol. Similar time-course changes of the concentrations suggest that the metabolism of dihydroconiferyl acetate is underlaid by the similar regulation to aromatic scent compounds. Dihydroconiferyl acetate and iso-eugenol occurred almost exclusively, implying that metabolism of the common precursors to each compound is selectively regulated in these plants. The branching of the biosynthetic pathway into dihydroconiferyl acetate and iso-eugenol is probably one of regulatory steps leading to scent diversity in P. axillaris lines.     

Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol

Petunia flower petals emit large amounts of isoeugenol, which has been shown to be synthesized by isoeugenol synthase (PhIGS1) from an ester of coniferyl alcohol, hypothesized to be coniferyl acetate. This paper describes the identification and characterization of a novel petunia gene encoding an enzyme belonging to the BAHD acyltransferase family whose expression correlates with isoeugenol biosynthesis. RNAi suppression of this gene results in inhibition of isoeugenol biosynthesis. Biochemical characterization of the protein encoded by this gene showed that it has acetyltransferase activity and is most efficient with coniferyl alcohol among the alcohol substrates tested. Overall, these data support the conclusion that coniferyl acetate is the substrate of isoeugenol synthase.     

Distribution of self-compatible and self-incompatible populations of Petunia axillaris (Solanaceae) outside Uruguay

Petunia axillaris occurs in temperate South America and consists of three allopatric subspecies: axillaris, parodii, and subandina. Previous studies have revealed that subsp. axillaris is self-incompatible (SI), subsp. parodii is self-compatible (SC) in Uruguay, and subsp. subandina is SC in Argentina. The SI/SC status over the entire distribution range is not completely understood, however. The objective of this study was to examine the overall SI/SC status of the respective subspecies in comparison with floral morphology. The results confirmed that subsp. parodii and subsp. subandina were SC throughout the distribution range, and that subsp. axillaris was also SC in Brazil and in most of the Argentinean territory. The SI P. axillaris occurs in the natural population only between 34 and 36°S, along the eastern shore of South America. The Brazilian and Uruguayan subsp. axillaris differed in SI/SC status and floral morphology. We discuss the cause of this difference.     

Reproductive Isolation in a Native Population of Petunia sensu Jussieu (Solanaceae)

Reproductive isolation was studied in four syntopic speciesof Petunia sensu Jussieu (Solanaceae) at a site in Rio Grandedo Sul State, Brazil. Reciprocal artificial crossing experimentsconfirmed that a genetic barrier exists between Petunia(P. axillarisand P. integrifolia) andCalibrachoa (C. parviflora and C. heterophylla),and also between C. parviflora andC. heterophylla . Petuniaaxillaris(white, nocturnally scented flower) is geneticallycompatible with the syntopic and allotopic P. integrifolia(coloured,unscented flower). Reproductive isolation appears to be maintainedby the two species having different pollinators: nocturnallyactive hawkmoths (Manduca contracta andM. diffissa subsp. petuniae)pollinate P. axillaris while a diurnally active bee (Hexanthedasp.) pollinates P. integrifolia. Flowers of P. integrifoliaexhibit diurnal opening and closing movements synchronous withthe activity period of the bee. Other than a probable nectarrobber (a carpenter bee, Xylocopa augusti), no insect visitedflowers of P. axillaris in the day. Amounts of floral nectarin P. axillaris and P. integrifolia were within the range ofhawkmoth- and bee-pollinated flowers, respectively. Reproductiveisolating mechanisms in the genus Petunia sensu Jussieu arediscussed. Copyright 2001 Annals of Botany Company Calibrachoa, hawkmoth, Petunia, Petunia axillaris, Petunia integrifolia, pollinator, reproductive isolation, Solanaceae     

Molecular phylogeny and DNA amplification fingerprinting of Petunia taxa

The relationship of five species of Petunia and ten cultivars of the cultivated petunia, Petunia x hybrida, were investigated using DNA-amplification fingerprinting (DAF). Reproducible banding profiles were obtained from P. parodii and P. axillaris DNA from different seed sources. In contrast, other petunias such as P. inflata, P. violacea and P. integrifolia produced variable fingerprints when different plants were examined. However, representative profiles of the variable Petunia taxa were obtained by bulking the leaf tissue from ten different individual plants. Each of ten octamer primers revealed polymorphic loci between taxa. Among a total of 201 bands produced, 146 (73%) loci were polymorphic and distinguished all species and cultivars. Phenetic and cluster analysis using DAF markers separated P. axillaris from P. parodii and distinguished between the violet-flowered species, P. inflata, P. violacea, and P. integrifolia. P. parodii grouped together with the monophyletic set of the ten cultivars of P. x hybrida examined, indicating that it had made a major contribution to the development of these cultivars. Cultivars were distributed within the dendograms by flower color. The results demonstrated the utility of DAF in establishing relationships among closely related species and cultivars of Petunia.     

Diversification in the South American Pampas: the genetic and morphological variation of the widespread Petunia axillaris complex (Solanaceae)

Understanding the spatiotemporal distribution of genetic variation and the ways in which this distribution is connected to the ecological context of natural populations is fundamental for understanding the nature and mode of intraspecific and, ultimately, interspecific differentiation. The Petunia axillaris complex is endemic to the grasslands of southern South America and includes three subspecies: P. a. axillaris, P. a. parodii and P. a. subandina. These subspecies are traditionally delimited based on both geography and floral morphology, although the latter is highly variable. Here, we determined the patterns of genetic (nuclear and cpDNA), morphological and ecological (bioclimatic) variation of a large number of P. axillaris populations and found that they are mostly coincident with subspecies delimitation. The nuclear data suggest that the subspecies are likely independent evolutionary units, and their morphological differences may be associated with local adaptations to diverse climatic and/or edaphic conditions and population isolation. The demographic dynamics over time estimated by skyline plot analyses showed different patterns for each subspecies in the last 100 000 years, which is compatible with a divergence time between 35 000 and 107 000 years ago between P. a. axillaris and P. a. parodii, as estimated with the IMa program. Coalescent simulation tests using Approximate Bayesian Computation do not support previous suggestions of extensive gene flow between P. a. axillaris and P. a. parodii in their contact zone.     

Metabolic regulation of floral scent in Petunia axillaris lines: biosynthetic relationship between dihydroconiferyl acetate and iso-eugenol

Aromatic scent-related compounds in flowers were comprehensively analyzed by high-performance liquid chromatography (HPLC) based on their absorption spectra to understand regulation of metabolism leading to floral scent diversity in Petunia axillaris lines. An unknown compound occurring at similar levels to scent compounds in some plant lines was identified to be dihydroconiferyl acetate. Based on the structure, dihydroconiferyl acetate is likely to be a biosynthetically closely related compound to aromatic scent compounds, especially iso-eugenol. Similar time-course changes of the concentrations suggest that the metabolism of dihydroconiferyl acetate is underlaid by the similar regulation to aromatic scent compounds. Dihydroconiferyl acetate and iso-eugenol occurred almost exclusively, implying that metabolism of the common precursors to each compound is selectively regulated in these plants. The branching of the biosynthetic pathway into dihydroconiferyl acetate and iso-eugenol is probably one of regulatory steps leading to scent diversity in P. axillaris lines.     

Infraspecific classification reflects genetic differentiation in the widespread Petunia axillaris complex: A comparison among morphological,ecological, and genetic patterns of geographic variation

Plants are subjected to natural selection during expansion of their geographic range or when facing changes in environmental conditions, which in turn may affect phenotypic diversity. Studies on geographic phenotypic variation provide insight into the evolutionary processes and have long contributed to better understanding of diversification within and among species. Petunia axillaris is widely distributed in temperate South America, occurring throughout the Pampas region. The current taxonomy of this species recognizes three allopatric subspecies, which occupy nearly adjacent territories, according to floral morphology: P. axillaris subsp. axillaris, P. axillaris subsp. parodii, and P. axillaris subsp. subandina. In this study, we sampled the three P. axillaris subspecies and used both molecular markers (from the plastid and nuclear genomes) and morphological measurements to investigate how genetic diversity and morphological variation correlate to ecological variables and the geographic context. We used different forms of Mantel tests (partial and correlograms) to investigate the geographic distribution patterns in distinct types of similarity/dissimilarity among the populations and their relationships. We also modeled the morphological variation in P. axillaris as a function of the genetic marker frequencies in the populations. We found that the morphological differences leading to the recognition of different subspecies of P. axillaris reflect historical processes of isolation and that adaptation to different ecological conditions faced by each lineage is perhaps not merely a consequence of phenotypic plasticity. These findings suggest that differences between subspecies could represent an incipient speciation stage.     

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.     

The composition and timing of flower odour emission by wild <Emphasis Type="Italic">Petunia axillaris</Emphasis> coincide with the antennal perception and nocturnal activity of the pollinator <Emphasis Type="Italic">Manduca sexta</Emphasis>

In the genus Petunia, distinct pollination syndromes may have evolved in association with bee-visitation (P. integrifolia spp.) or hawk moth-visitation (P. axillaris spp). We investigated the extent of congruence between floral fragrance and olfactory perception of the hawk moth Manduca sexta. Hawk moth pollinated P. axillaris releases high levels of several compounds compared to the bee-pollinated P. integrifolia that releases benzaldehyde almost exclusively. The three dominating compounds in P. axillaris were benzaldehyde, benzyl alcohol and methyl benzoate. In P. axillaris, benzenoids showed a circadian rhythm with an emission peak at night, which was absent from P. integrifolia. These characters were highly conserved among different P. axillaris subspecies and P. axillaris accessions, with some differences in fragrance composition. Electroantennogram (EAG) recordings using flower-blends of different wild Petunia species on female M. sexta antennae showed that P. axillaris odours elicited stronger responses than P. integrifolia odours. EAG responses were highest to the three dominating compounds in the P. axillaris flower odours. Further, EAG responses to odour-samples collected from P. axillaris flowers confirmed that odours collected at night evoked stronger responses from M. sexta than odours collected during the day. These results show that timing of odour emissions by P. axillaris is in tune with nocturnal hawk moth activity and that flower-volatile composition is adapted to the antennal perception of these pollinators.     

Nucleus substitution between Petunia species using gamma ray-induced androgenesis

Summary The ovaries of two different Petunia species: Petunia hybrida (hort) and Petunia parodii (Steere) were irradiated with -ray doses ranging from 50 to 1,000 Gy before pollination. Seed setting occurred after 4 days preculture on a non-sterile medium. Ovaries transformed into fruits were then cultivated aseptically with the following results: (1) -ray doses ranging from 200 to 1,000 Gy led to the development of two types of plants: haploids 2n=x=7 and overdiploids 2n>2x=14. (2) The androgenetic origin of haploids was ascertained by using genetic markers. The origin of overdiploids is discussed. (3) Androgenetic haploids contained the chloroplasts of the irradiated female parent. No visible change of cp DNA patterns was observed after irradiation. (4) The four possible androgenetic events were successfully obtained between the two Petunia species: hybrida haploids with hybrida or parodii cytoplasm, and parodii haploids with parodii or hybrida cytoplasm.     

Location of structural genes for glucose phosphate isomerase and for leucyl aminopeptidase on chromosome VII of Petunia

Summary A gene termed gpiB, coding for one of the two isoenzyme zones of glucose phosphate isomerase in Petunia, has been mapped to a locus on chromosome VII by means of linkage to the marker An4, and by an allelic dosage effect on enzyme activity in trisomics. The high degree of linkage of electrophoretic alleles of gpiB to the pollen colour allele pair An4/an4, as demonstrated in the ancestral species, P. axillaris s.l. and P. integrifolia s.l., has been conserved in all cultivars of P. hybrida investigated. Another gene, coding for the enzyme leucyl-aminopeptidase could also be mapped to chromosome VII and the gene order An4 — lapB — gpiB determined. Apparently, distribution of lapB alleles is not related to the hybrid descent of P. hybrida.     

Biotransformation of isoeugenol catalyzed by growing cells of Pseudomonas putida

Abstract

Growing cells of Pseudomonas putida transformed isoeugenol after 5 days of incubation to give mainly vanillin, eugenol, 4-(E)-(3-hydroxyprop-1-enyl)-2-methoxyphenol and the dimeric molecule (+)-4-[2,3-dihydro-7-methoxy-3-methyl-5-(E)-(1-propenyl)-2-benzofuranyl]-2-methoxyphenol (licarin A). The formation of the latter compound from isoeugenol by biotransformation with P. putida is reported here for the first time.