Hisashi Kokubun

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.

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.

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.

Differences in the floral anthocyanin content of red petunias and Petunia exserta.

In order to resolve a conflict between previous papers regarding the floral anthocyanins of red flowers of Petunia exserta, a naturally occurring species, the HPLC profile of this species was compared with that of commercial red garden petunias. Both HPLC profiles extremely superficially resemble each other in terms of relative amounts and retention times of the major anthocyanins. However, co-elution on HPLC of the mixed sample resulted in clear separation of the components. Three major anthocyanins in red petunias were determined to be cyanidin 3-sophoroside, cyanidin 3-glucoside and peonidin 3-glucoside, which exhibited similar behaviors on HPLC to delphinidin 3-glucoside. delphinidin-3-rutinoside and petunidin 3-rutinoside, respectively, the major floral anthocyanins of P. exserta.

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.

Insertion and excision of a transposable element governs the red floral phenotype in commercial petunias

Commercial cultivars of Petunia hybrida with red flowers (red petunias) accumulate cyanidin 3-glucoside as a main floral anthocyanin pigment. The conversion of anthocyanidin 3-glucosides to anthocyanidin 3-rutinosides is catalyzed by a UDP-rhamnose: anthocyanidin 3-glucoside-rhamnosyltransferase (RT). In red petunias, the RT gene is known to be disrupted by the insertion of a transposable element (dTph3). We have cloned the dTph3-like element (called dTph3-C) from a red petunia. Unlike dTph3, dTph3-C harbored a perfect terminal inverted repeat. In addition, an excision product (so-called “footprint”) of dTph3/dTph3-C was found in another red petunia. The RT transcripts harboring this footprint could not produce the RT enzyme because a stop codon was created in the footprint sequence. The genotypes of the 42 commercial petunias which exhibit different anthocyanin pigmentation were determined by multiplex PCR. In this technique, the amplified products from normal, dTph3/dTph3-C-inserted, and footprint-retaining RT genes can be separated from one another. Our results indicate that the red-floral phenotype of commercial petunias is governed by insertion and excision events of a transposable element in the coding region of the RT gene.

Nuclear DNA content in the genus Hepatica (Ranunculaceae)

Using flow cytometry, we measured the nuclear DNA contents of all known taxa in Hepatica. Nuclear DNA content of Hepatica falconeri (diploid, crenate leaf lobes) was significantly lower than that of diploid entire species. Among the tetraploid species, crenate species had lower DNA contents than the entire taxon H. nobilis var. pubescens. The DNA content of the tetraploid species was more than double that of the diploid species among the same leaf-type groups.

Floral coloration and pigmentation in Calibrachoa cultivars

Summary The ¯oral pigment pro®les of twenty in Calibrachoa cultivars were determined and compared with the pro®les of selected Petunia cultivars. Calibrachoas could be divided into two groups based on high (177±871.mgg±1 FW) and low 2±9.mg g±1 FW) carotenoid content; the groups were represented by yellow and white ¯owers, respectively. In addition to yellow cultivars, the high carotenoid content group included vivid-red and vivid-purplish-red cultivars that were characterized by a low ¯oral pH of 4.5±4.8 and accumulation of delphinidin and petunidin, respectively, as the major ¯oral anthocyanidin. In addition to cultivars with white ¯owers, the low carotenoid content group contained vivid-purple and vivid-red-purple cultivars that were characterized by higher (pH 5.4±5.7) and intermediate (pH 4.8±5.3) corolla pHs, respectively, and accumulation of malvidin. The strategies of calibrachoas and petunias for expressing yellow and red ¯owers were found to differ. For yellow ¯owers, calibrachoas use carotenoids, whereas petunias use ¯avonols plus carotenoids. For red ¯owers, calibrachoas use carotenoids, delphinidin and lower corolla pH, whereas petunias use cyanidin and lower corolla pH.