Toshio Ando

Collaborative non-self recognition system in S-RNase-based self-incompatibility.

Dissecting Self-Incompatibility Although the pollen may be available for a flower to fertilize itself, molecular determinants on the pollen and the pistil prevent inbreeding in a process termed self-incompatibility. In the Petunia self-incompatibility, if male determinants (F-box proteins) on pollen are recognized by a female ribonuclease determinant on the pistil, the pollen tube is killed when its ribosomal RNA is digested. Outcrossed fertilizations can occur because of allelic diversity in the female that fails to recognize its male counterparts; however, the genetic diversity of the ribonuclease gene is greater than that of the known F-box gene. Kubo et al. (p. 796; see the Perspective by Indriolo and Goring) have discovered that there are several related F-box genes in Petunia, each of which brings its own allelic diversity to bear—thus, increasing the variety of potential mating partners. A highly diverse set of (male) pollen-determinant proteins in Petunia recognizes non-self (female) pistil determinants. Self-incompatibility in flowering plants prevents inbreeding and promotes outcrossing to generate genetic diversity. In Solanaceae, a multiallelic gene, S-locus F-box (SLF), was previously shown to encode the pollen determinant in self-incompatibility. It was postulated that an SLF allelic product specifically detoxifies its non-self S-ribonucleases (S-RNases), allelic products of the pistil determinant, inside pollen tubes via the ubiquitin–26S-proteasome system, thereby allowing compatible pollinations. However, it remained puzzling how SLF, with much lower allelic sequence diversity than S-RNase, might have the capacity to recognize a large repertoire of non-self S-RNases. We used in vivo functional assays and protein interaction assays to show that in Petunia, at least three types of divergent SLF proteins function as the pollen determinant, each recognizing a subset of non-self S-RNases. Our findings reveal a collaborative non-self recognition system in plants.

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.

Complete amino acid sequence of boar protamine

Abstract The complete amino acid sequence of boar protamine was determined. In order to specify cleaving points for enzymatic fragmentation, two S-alkyl derivatives of the protamine were used. 1. The sequences other than arginine stretches were determined by analysis of tryptic peptides from the S-carboxymethyl-protamine. The sequence of the central arginine-clustered region was determined by stepwise degradation from the C-terminus of the thermolysin core-fragment with carboxypeptidase A and B, and then with acid carboxy-peptidase. 2. S-Methylation of cysteine residues of the protamine was found to introduce a new point for thermolysin and chymotryptic cleavage. Thus, thermolysin cleavage of the chymotryptic core-fragment resulted in oligo-arginine peptides with methylcysteine at the N-terminus, by which the sequence of arginine-clusters was confirmed. 3. The complete sequence was deduced by overlap of these sequenced peptides, which is: Ala-Arg-Tyr-Arg-Cys2-Arg-Ser-His-Ser-Arg-Ser-Arg-Cys-Arg-Pro-Arg4-Cys-Arg6-Cys2-Pro-Arg5-Ala-Val-Cys2- Arg2-Tyr-Thr-Val-Ile-Arg-Cys-Arg2Cys. Boar protamine, as well as bull protamine, is composed of the less basic amino- and carboxy-terminal domains and the central arginine-clustered domain, and 80% homology was found between boar and bull protamines.

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.

Tandemly arranged chalcone synthase A genes contribute to the spatially regulated expression of siRNA and the natural bicolor floral phenotype in Petunia hybrida

The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event.

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.

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed intrageneric classifications were evaluated. The phylogenetic tree showed that (1) sectionsArguta, Cissifolia, Lithocarpa, Macrantha, Palmata, Spicata, Tataricum, Trifoliata sensu Ogata (1967: Bull. Tokyo Univ. Forests 63: 89–206) were monophyletic groups respectively, (2) sectionsCampestria, Goniocarpa, Platanoidea sensu Ogata (1967) were polyphyletic respectively, and (3) sectionsDistyla andParviflora formed a sister group. An average of estimated nucleotide substitution rates of Acer chloroplast DNA was calculated as 7.9×10−11±1.4×10−11 nucleotide substitutions par site par year, which coincides well with previously reported rates of perennial plants. Divergence eras of eastern Asia and North American species in both sectionsSpicata andRubra were estimated to be late Miocene. In consideration with previous data, multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distribution.