Eduardo Marchesi

Floral anthocyanins in wild taxa of Petunia (Solanaceae)

The flowers of 20 native taxa of Petunia (Solanaceae) were investigated by HPLC for the occurrence of anthocyanins. The investigation revealed the presence of at least 24 anthocyanins in their flowers, of which 18 known anthocyanins isolated from the flowers of P. exserta, P. guarapuavensis, P. integrifolia, P. occidentalis, and P. reitzii were fully identified by chemical and spectral methods to be 3-glucoside of delphinidin; 3-rutinosides of cyanidin, delphinidin, and petunidin; 3-rutinoside-5-glucosides, 3-trans and -cis-p-coumaroylrutinoside-5-glucosides, and 3-trans-caffeoylrutinoside-5-glucosides of delphinidin, petunidin, and malvidin; and 3-transcaffeoylglucosyl-trans-(caffeoyl or p-coumaroyl) rutinoside-5-glucosides of malvidin. Six novel anthocyanins were isolated from the flowers of P. occidentalis, and their structures were identified to be 3-glucosyl p-coumaroylrutinoside-5-glucosides and 3-glucosylcaffeoylrutinosides of petunidin and malvidin, and also 3-caffeoylglucosylcaffeoylrutinoside-5-glucoside and 3-caffeoylglucosyl p-coumaroylrutinoside-5-glucoside of petunidin. Out of the six pigments, petunidin 3-glucosyl p-coumaroylrutinoside-5-glucoside was unambiguously determined by spectral methods to be petunidin 3-O-[6-O-(4-O-(4-O-(β-d-glucopyranosyl)-trans-p-coumaroyl)-α-l-rhamnopyranosyl)-β-d-glucopyranoside]- 5-O-[β-d-glucopyranoside]. The 20 native taxa of Petunia could be placed into four groups (A, B, C, and D) with one further into five subgroups (D1–D5) regarding their constituents and contents of major anthocyanins and also their pigment biosynthesis with respect to the blocks or inhibitors of the hydroxylation, glucosylation, and acylation reactions in them. The use of anthocyanins as taxonomic markers in the genus Petunia is discussed in relation to the flower colour and possible pollination vectors.

Duplication of the S-locus F-box gene is associated with breakdown of pollen function in an S-haplotype identified in a natural population of self-incompatible Petunia axillaris.

We previously identified both self-incompatible and self-compatible plants in a natural population of self-incompatible Petunia axillaris subsp. axillaris, and found that all the self-compatible plants studied carried either SC1- or SC2-haplotype. Genetic crosses showed that SC2 was identical to S17 identified from another natural population of P. axillaris, except that its pollen function was defective, and that the pollen-part mutation in SC2 was tightly linked to the S-locus. Recent identification of the S-locus F-box gene (SLF) as the gene that controls pollen specificity in S-RNase-based self-incompatibility has prompted us to examine the molecular basis of this pollen-part mutation. We cloned and sequenced the S17-allele of SLF of P.axillaris, named PaSLF17, and found that SC2SC2 plants contained extra restriction fragments that hybridized to PaSLF17 in addition to all of those observed in S17S17 plants. Moreover, these additional fragments co-segregated with SC2. We used the SC2-specific restriction fragments as templates to clone an allele of PaSLF by PCR. To determine the identity of this allele, named PaSLFx, primers based on its sequence were used to amplify PaSLFalleles from genomic DNA of 40 S-homozygotes of P. axillaris, S1S1 through S40S40. Sequence comparison revealed that PaSLFx was completely identical with PaSLF19 obtained from S19S19. We conclude that the S-locus of SC2 contained both S17-allele and the duplicated S19-allele of PaSLF. SC2 is the first naturally occurring pollen-part mutation of a solanaceous species that was shown to be associated with duplication of the pollen S. This finding lends support to the proposal, based on studies of irradiation-generated pollen-part mutants of solanaceous species, that duplication, but not deletion, of the pollen S, causes breakdown of pollen function.

Emission Mechanism of Floral Scent in Petunia axillaris

The mechanism of floral scent emission was studied in Petunia axillaris, a plant with a diurnal rhythm of scent output. The emission rate of each volatile compound oscillated in synchrony with its endogenous concentration, so that the intensity of the floral scent appeared to be determined by the endogenous concentrations. The composition of major volatiles in the flower tissue and the flower headspace showed characteristic differences. A negative correlation was found between the boiling points of the volatile compounds and the ratio of their emitted and endogenous concentrations, indicating that the composition of the floral scent depends directly on the endogenous composition of the volatile compounds. We conclude that in P. axillaris, the physiological regulation of floral scent emission operates not in the vaporization process but in the control of the endogenous concentrations of volatiles through biosynthesis and metabolic conversion.

Breakdown of self-incompatibility in a natural population of Petunia axillaris (Solanaceae) in Uruguay containing both self-incompatible and self-compatible plants

Abstract Many members of the Solanaceae display a type of gametophytic self-incompatibility which is controlled by a single multiallelic locus, called the S-locus. From our previous survey of more than 100 natural populations of Petunia axillaris (a solanaceous species) in Uruguay, we had found that the majority of the populations of subspecies axillaris were comprised of virtually all self-incompatible individuals. The rest were ”mixed populations” which contained mostly self-incompatible and some self-compatible individuals. In this study, we examined the self-incompatibility behavior and determined the S-genotypes of 33 plants raised from seeds obtained from one such mixed population, designated U1. We found that 30 of the 33 plants (designated U1–1 through U1–33) were self-incompatible and a total of 18 different S-alleles were represented. To determine the S-genotypes of the three self-compatible plants (U1–2, U1–16, and U1–22) and the possible causes for the breakdown of their self-incompatibility, we carried out reciprocal crosses between each of them and each of the 18 S-homozygotes (S1S1 through S18S18) obtained from bud-selfed progeny of 14 of the 30 self-incompatible plants. For U1–2 and U1–16, we also carried out additional crosses with U1–25 (with S1S13 genotype) and an S13S15 plant (obtained from a cross between an S13-homozygote and an S15-homozygote), respectively. Based on all the pollination results and analysis of the production of S-RNases, products of S-alleles in the pistil, we determined the S-genotypes of U1–2, U1–16, and U1–22, and propose that the breakdown of self-incompatibility in these three plants is caused by suppression of the production of S13-RNase from the S13-allele they all carry. We have termed this phenomenon ”stylar-part suppression of an S-allele” or SPS.

Breakdown of Self-Incompatibility in a Natural Population of Petunia axillaris Caused by Loss of Pollen Function

Although Petunia axillaris subsp.axillaris is described as a self-incompatible taxon, some of the natural populations we have identified in Uruguay are composed of both self-incompatible and self-compatible plants. Here, we studied the self-incompatibility (SI) behavior of 50 plants derived from such a mixed population, designated U83, and examined the cause of the breakdown of SI. Thirteen plants were found to be self-incompatible, and the other 37 were found to be self-compatible. A total of 14 S-haplotypes were represented in these 50 plants, including two that we had previously identified from another mixed population, designated U1. All the 37 self-compatible plants carried either an SC1 - or anSC2 -haplotype.SC1 SC1andSC2 SC2homozygotes were generated by self-pollination of two of the self-compatible plants, and they were reciprocally crossed with 40 self-incompatible S-homozygotes (S1 S1throughS40 S40) generated from plants identified from three mixed populations, including U83. TheSC1 SC1homozygote was reciprocally compatible with all the genotypes examined. The SC2 SC2homozygote accepted pollen from all but theS17 S17homozygote (identified from the U1 population), but theS17 S17homozygote accepted pollen from theSC2 SC2homozygote. cDNAs encoding SC2- and S17-RNases were cloned and sequenced, and their nucleotide sequences were completely identical. Analysis of bud-selfed progeny of heterozygotes carrying SC1 orSC2 showed that the SI behavior of S C1 and S C2 was identical to that of S C1 andS C2 homozygotes, respectively. All these results taken together suggested that the S C2 -haplotype was a mutant form of the S 17 -haplotype, with the defect lying in the pollen function. The possible nature of the mutation is discussed.

Effect of Temperature on the Floral Scent Emission and Endogenous Volatile Profile of Petunia axillaris

The floral scent emission and endogenous level of its components in Petunia axillaris under different conditions (20, 25, 30, and 35 °C) were investigated under the hypothesis that floral scent emission would be regulated by both metabolic and vaporization processes. The total endogenous amount of scent components decreased as the temperature increased, the total emission showing a peak at 30 °C. This decrease in endogenous amount was compensated for by increased vaporization, resulting in an increase of floral scent emission from 20 °C to 30 °C. The ambient temperature differently and independently influenced the metabolism and vaporization of the scent compounds, and differences in vapor pressure among the scent compounds were reduced as the temperature increased. These characteristics suggest the operation of an unknown regulator to change the vaporization of floral scent.

Phylogenetic analysis of the genus Petunia (Solanaceae) based on the sequence of the Hf1 gene

Polymerase chain reaction fragment length polymorphisms and nucleotide sequences for a cytochrome P450 gene encoding flavonoid-3′,5′-hydroxylase, Hf1, were studied in 19 natural taxa of Petunia. Natural Petunia taxa were classified into six groups based on major insertion or deletion events that occurred only in intron II of the locus. The maximum parsimony method was used to calculate strict consensus trees based on nucleotide sequences in selected regions of the Hf1 locus. Petunia taxa were divided into two major clades in the phylogenetic trees. Petuniaaxillaris (including three subspecies), P. exserta, and P. occidentalis formed a clade with 100% bootstrap support. This clade is associated with a consistently inflexed pedicel, self-compatibility in most taxa, and geographical distribution in southern and western portions of the genus range. The other clade, which comprised the remainder of the genus is, however, less supported (up to 71% bootstrap); it is characterized by a deflexed pedicel in the fruiting state (except P. inflata), self-incompatibility, and a northeastern distribution. A nuclear gene, Hf1, seems to be a useful molecular marker for elucidating the phylogeny of the genus Petunia when compared with the nucleotide sequence of trnK intron of chloroplast DNA.

Floral scent diversity is differently expressed in emitted and endogenous components in Petunia axillaris lines.

• Background and Aims Among the subspecies of Petunia axillaris are various lines emitting sensorially different scents. Analysis of variations in floral scent among genetically close individuals is a powerful approach to understanding the mechanisms for generating scent diversity. • Methods Emitted and endogenous components were analysed independently to gain information about evaporation and endogenous production in 13 wild lines of P. axillaris. A dynamic headspace method was used to collect emitted components. Endogenous components were extracted with solvent. Both of these sample types were subjected to quantitative and qualitative analysis by gas chromatography (GC)–flame ionization detector (FID) and GC–mass spectrometry (MS). • Key Results and Conclusions Whereas the profiles of emitted compounds showed qualitative homogeneity, being mainly composed of methyl benzoate with quantitative variation, the profiles of endogenous compounds showed both qualitative and quantitative variation. A negative correlation was found between the evaporation ratio and boiling point of each compound examined. Lower boiling point compounds were strongly represented in the emitted component, resulting in the reduction of qualitative variation in floral scent. In conclusion, floral scent diversity results from variation in both the endogenous production and the evaporation rate of the individual volatile compounds.

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.

Three groups of species in Petunia sensu Jussieu (Solanaceae) inferred from the intact seed morphology

The intact seed surface morphology in 45 taxa of Petunia sensu Jussieu native to South America (Petunia sensu Wijsman plus Calibrachoa) was compared under scanning electron microscopy. The existence of three groups of species, differentiated in terms of seed morphology, was revealed as follows: (1) all species of Petunia sensu Wijsman, having coarse wavy middle lamellae and anticlinal walls; (2) Calibrachoa parviflora and C. pygmaea, having fine wavy middle lamellae embedded in straight anticlinal walls; and (3) the other species of Calibrachoa, having straight middle lamellae and anticlinal walls. Close relationships between seed morphology and the other characteristics observable in the groups of species are discussed.