Inhibition of post-transcriptional gene silencing of chalcone synthase genes in petunia picotee petals by fluacrypyrim.

In petals of picotee petunia (Petunia hybrida) cultivars, margin-specific post-transcriptional gene silencing (PTGS) of chalcone synthase A (CHSA) inhibits anthocyanin biosynthesis, resulting in marginal white tissue formation. In this study, we found that a low molecular mass compound, fluacrypyrim, inhibits PTGS of CHSA, and we explored the site-specific PTGS mechanism of operation. Fluacrypyrim treatment abolished the picotee pattern and eliminated site-specific differences in the levels of anthocyanin-related compounds, CHSA expression, and CHSA small interfering RNA (siRNA). In addition, fluacrypyrim abolished the petunia star-type pattern, which is also caused by PTGS of CHSA. Fluacrypyrim treatment was effective only at the early floral developmental stage and predominantly eliminated siRNA derived from CHS genes; i.e. siRNA derived from other genes remained at a comparable level. Fluacrypyrim probably targets the induction of PTGS that specifically operates for CHS genes in petunia picotee flowers, rather than common PTGS maintenance mechanisms that degrade mRNAs and generate siRNA. Upon treatment, the proportion of colored tissue increased due to a shift of the border between white and colored sites toward the margin in a time- and dose-dependent manner. These findings imply that the fluacrypyrim-targeted PTGS induction is completed gradually and its strength is attenuated from the margins to the center of petunia picotee petals.

Heredity of flake- and stripe-variegated traits and their introduction into Japanese day-neutral winter-flowering sweet pea (Lathyrus odoratus L.) cultivars.

Sweet pea (Lathyrus odoratus L.) is a major cut flower in Japan, generally grown in greenhouses in winter to spring. The wild-type sweet pea is a long-day summer-flowering plant. The day-neutral winter-flowering ability, which allows cut-flower production in Japan, is a recessive phenotype that emerged by spontaneous mutation. Although Japanese winter-flowering cultivars and additionally spring-flowering cultivars, which have semi-long-day flowering ability generated by crossing the winter- and summer-flowering cultivars, have superior phenotypes for cut flowers, they have limited variation in color and fragrance. In particular, variegated phenotypes do not appear in modern winter- and spring-flowering cultivars, only in summer-flowering cultivars. We try to expand the phenotypic diversity of Japanese cut flower cultivars. In the processes, we introduced the variegated phenotypes by crossing with summer-flowering cultivars, and succeeded in breeding some excellent cultivars. During breeding, we analyzed the segregation ratios and revealed the heredity of the phenotypes. Here we review the heredity of these variegated phenotypes and winter-flowering phenotypes and their related genes. We also describe how we introduced the trait into winter-flowering cultivars, tracing their pedigrees to show both phenotypes and genotypes of the progeny at each generation. This knowledge is useful for the efficient breeding of new variegated cultivars.

Generation of blue chrysanthemums by anthocyanin B-ring hydroxylation and glucosylation and its coloration mechanism.

Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)-glucose:anthocyanin 3',5'-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3'5'H. Newly synthesized 3',5'-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed "copigmentation," with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.

Red-purple flower color and delphinidin-type pigments in the flowers of Pueraria lobata (Leguminosae).

A previously undescribed acylated anthocyanin was extracted from the red-purple flowers of Pueraria lobata with 5% HOAc-H2O, and determined to be petunidin 3-O-(ß-glucopyranoside)-5-O-[6-O-(malonyl)-ß-glucopyranoside], by chemical and spectroscopic methods. In addition, two known acylated anthocyanins, delphinidin 3-O-(ß-glucopyranoside)-5-O-[6-O-(malonyl)-ß-glucopyranoside] and malvidin 3-O-(ß-glucopyranoside)-5-O-[6-O-(malonyl)-ß-glucopyranoside] were identified. Delphinidin 3,5-di-glucoside, petunidin 3,5-di-glucoside, and malvidin 3,5-di-glucoside, have been known as major components of P. lobata in the former study. However, malonyl esters amounts were detected over 10 times compared with non-malonyl esters amounts. In those anthocyanins the most abundant anthocyanin was petunidin 3-O-(ß-glucopyranoside)-5-O-[6-O-(malonyl)-ß-glucopyranoside] in total flowers. On the visible absorption spectral curve of fresh red-purple petals, one characteristic absorption maximum was observed at 520 nm, which is similar to those of flowers containing pelargonidin derivatives. In contrast, the absorption spectral curve of old violet petals was observed at 500(sh), 536, 564(sh), and 613(sh) nm, which are similar to those of violet flowers containing delphinidin-type pigments. Pressed juices of both fresh red-purple petals and old violet petals had pH5.2 and 5.5 respectively, and had the same flavonoid constitution. Crude fresh red-purple petal pigments extracted by pH 2.2 and pH 5.2 buffers exhibited the same color and spectral curves as fresh red-purple petals and old violet petals, respectively. Moreover, in a cross-TLC experiment of crude extracted pigments, red-purple color was exhibited by the anthocyanin region and the crossed region of anthocyanins and isoflavone. Thus, it may be assumed that the unusually low pH in the vacuole of fresh petals plays an important role to form red-purple flower color against weak acidic pH in the vacuole of old violet P. lobata petals.

Characteristics of green-blue fluorescence generated from the adaxial sides of leaves of tree species.

We discovered that some tree species have leaves whose adaxial sides show bright green-blue fluorescence upon exposure to ultraviolet irradiation. In total, 141 native Japanese species belonging to 47 families were analyzed, and the brightness of the leaf fluorescence, represented by the L* values (Lab color space) of the pictures, was evaluated. The species possessing the brightest fluorescent leaves, with L* > 50, were Camellia japonica, Camellia sasanqua, and Cleyera japonica of Theaceae, Osmanthus heterophyllus and Ligustrum japonicum of Oleaceae, Aucuba japonica of Garryaceae, and Trochodendron aralioides of Trochodendraceae. These species are propagated by pollination or seed dispersion by birds, except T. aralioides. The fluorescence was specifically observed in the cuticle tissues of the epidermal cells, indicating that the fluorescence is a signal to other organisms that can perceive the fluorescence under natural light. Species possessing the bright leaves represented 5% of the total species tested, while species possessing dark leaves, with L* ≤ 40, represented 88.6%. We deduce that the fluorescence enables the organisms to easily distinguish the minority species possessing bright leaves from the surrounding plants, which were mostly trees species with dark leaves. The structure of A. japonica var. borealis, in which dark leaves only surround its fruits while the rest of the tree is covered by bright leaves, may be useful to signal the presence of fruits to the organisms. We hypothesize that the fluorescence contributes to the propagation of the tree species by helping birds to distinguish these particular trees and/or locate the fruits.

Flavone C-glucosides responsible for yellow pigmentation induced by low temperature in bracts of Zantedeschia aethiopica.

We aimed to identify the main compounds responsible for low temperature-induced yellow pigmentation of the bracts of Zantedeschia aethiopica 'Wedding March'. On the basis of the area ratios estimated from absorbance at 400 nm in HPLC analyses, we identified two flavonoids, isoorientin and swertiajaponin, as such compounds. We also identified two additional flavonoids, isovitexin and swertisin, which do not contribute considerably to the yellow pigmentation. Flavonoids of Zantedeschia bracts seem to belong to the class of flavone C-glycosides.

Anthocyanins in the bracts of Curcuma species and relationship of the species based on anthocyanin composition.

Five anthocyanins, delphinidin 3-O-rutinoside, cyanidin 3-O-rutinoside, petunidin 3-O-rutinoside, malvidin 3-O-glucoside and malvidin 3-O-rutinoside, were identified. Three anthocyanins, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside and pelargonidin 3-O-rutinoside, were putatively identified based on C18 HPLC retention time, absorption spectrum, including λmax, and comparisons with those of corresponding standard anthocyanins, as the compounds responsible for the pink to purple-red pigmentation of the bracts of Curcuma alismatifolia and five related species. Cluster analysis based on four major anthocyanins formed two clusters. One consisted of only one species, C. alismatifolia, and the other consisted of five. Each cluster further formed sub-clusters depending on either species or habitats.

Co-modification of class B genes TfDEF and TfGLO in Torenia fournieri Lind. alters both flower morphology and inflorescence architecture.

The class B genes DEFICIENS (DEF)/APETALA3 (AP3) and GLOBOSA (GLO)/PISTILLATA (PI), encoding MADS-box transcription factors, and their functions in petal and stamen development have been intensely studied in Arabidopsis and Antirrhinum. However, the functions of class B genes in other plants, including ornamental species exhibiting floral morphology different from these model plants, have not received nearly as much attention. Here, we examine the cooperative functions of TfDEF and TfGLO on floral organ development in the ornamental plant torenia (Torenia fournieri Lind.). Torenia plants co-overexpressing TfDEF and TfGLO showed a morphological alteration of sepals to petaloid organs. Phenotypically, these petaloid sepals were nearly identical to petals but had no stamens or yellow patches like those of wild-type petals. Furthermore, the inflorescence architecture in the co-overexpressing torenias showed a characteristic change in which, unlike the wild-types, their flowers developed without peduncles. Evaluation of the petaloid sepals showed that these attained a petal-like nature in terms of floral organ phenotype, cell shape, pigment composition, and the expression patterns of anthocyanin biosynthesis-related genes. In contrast, torenias in which TfDEF and TfGLO were co-suppressed exhibited sepaloid petals in the second whorl. The sepaloid petals also attained a sepal-like nature, in the same way as the petaloid sepals. The results clearly demonstrate that TfDEF and TfGLO play important cooperative roles in petal development in torenia. Furthermore, the unique transgenic phenotypes produced create a valuable new way through which characteristics of petal development and inflorescence architecture can be investigated in torenia.

A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)-related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio-temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3-MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale-colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.

Sequence analysis of the genome of carnation (Dianthus caryophyllus L.).

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. 'Francesco' was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568,887,315 bp, consisting of 45,088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16,644 bp and 60,737 bp, respectively, and the longest scaffold was 1,287,144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼ 98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.

Use of image analysis to estimate anthocyanin and UV-excited fluorescent phenolic compound levels in strawberry fruit.

Strawberry is rich in anthocyanins, which are responsible for the red color, and contains several colorless phenolic compounds. Among the colorless phenolic compounds, some, such as hydroxycinammic acid derivatives, emit blue-green fluorescence when excited with ultraviolet (UV) light. Here, we investigated the effectiveness of image analyses for estimating the levels of anthocyanins and UV-excited fluorescent phenolic compounds in fruit. The fruit skin and cut surface of 12 cultivars were photographed under visible and UV light conditions; colors were evaluated based on the color components of images. The levels of anthocyanins and UV-excited fluorescent compounds in each fruit were also evaluated by spectrophotometric and high performance liquid chromatography (HPLC) analyses, respectively and relationships between these levels and the image data were investigated. Red depth of the fruits differed greatly among the cultivars and anthocyanin content was well estimated based on the color values of the cut surface images. Strong UV-excited fluorescence was observed on the cut surfaces of several cultivars, and the grayscale values of the UV-excited fluorescence images were markedly correlated with the levels of those fluorescent compounds as evaluated by HPLC analysis. These results indicate that image analyses can select promising genotypes rich in anthocyanins and fluorescent phenolic compounds.

Metabolome profiling of floral scent production in Petunia axillaris.

Emission of floral scent benzenoid/phenylpropanoid compounds in Petunia axillaris increases significantly at night, a change that is primarily determined by the endogenous concentration of these compounds in the corolla. Among wild type P. axillaris plants, there are lines that emit different amounts of scent. To understand how the nocturnal rhythm of floral scent concentrations is controlled, the concentration profiles of metabolites in the scent biosynthetic pathway in two lines of P. axillaris, a strongly scented line and a weakly scented line, are reported. In the strongly scented line, the concentration of a series of compounds from glucose-6-phosphate (G6P) to the scent compounds changed synchronously. In the weakly scented lines, the concentrations of some metabolites including 6-phosphogluconate (6PG) and downstream metabolites of shikimic acid were remarkably lower, suggesting a reduction in metabolism of G6P to 6PG and the metabolism of shikimic acid in the weakly scented line. Nocturnal increases in the concentrations of sucrose, fructose, and glucose were not found in strongly scented line. Nocturnal increases in concentrations of S-adenosylhomocysteine (SAH) and methionine and reductions in the concentrations of S-adenosylmethionine (SAM), a methylation donor to benzenoid-skeletons, were observed only in strongly scented line. It is concluded that the biosynthetic regulation of each step from G6P to the volatile scent benzenoids is performed by, at least in part, concentrations of substrates, and the regulation also affects concentrations of SAM cycle compounds.

An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3'-hydroxylase gene.

The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3'H, the gene for flavonoid 3'-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.

Reverted glutathione S-transferase-like genes that influence flower color intensity of carnation (Dianthus caryophyllus L.) originated from excision of a transposable element.

A glutathione S-transferase-like gene, DcGSTF2, is responsible for carnation (Dianthus caryophyllus L.) flower color intensity. Two defective genes, DcGSTF2mu with a nonsense mutation and DcGSTF2-dTac1 containing a transposable element dTac1, have been characterized in detail in this report. dTac1 is an active element that produces reverted functional genes by excision of the element. A pale-pink cultivar 'Daisy' carries both defective genes, whereas a spontaneous deep-colored mutant 'Daisy-VPR' lost the element from DcGSTF2-dTac1. This finding confirmed that dTac1 is active and that the resulting reverted gene, DcGSTF2rev1, missing the element is responsible for this color change. Crosses between the pale-colored cultivar '06-LA' and a deep-colored cultivar 'Spectrum' produced segregating progeny. Only the deep-colored progeny had DcGSTF2rev2 derived from the 'Spectrum' parent, whereas progeny with pale-colored flowers had defective forms from both parents, DcGSTF2mu and DcGSTF2-dTac1. Thus, DcGSTF2rev2 had functional activity and likely originated from excision of dTac1 since there was a footprint sequence at the vacated site of the dTac1 insertion. Characterizing the DcGSTF2 genes in several cultivars revealed that the two functional genes, DcGSTF2rev1 and DcGSTF2rev2, have been used for some time in carnation breeding with the latter in use for more than half a century.

Pigment accumulation and transcription of LhMYB12 and anthocyanin biosynthesis genes during flower development in the Asiatic hybrid lily (Lilium spp.).

Anthocyanin biosynthesis is often regulated by MYB transcription factors that are classified into AN2 and C1 subgroups. The AN2 subgroup regulates the late genes in the anthocyanin biosynthesis pathway of eudicots, whereas the C1 subgroup controls both early and late genes in monocots. Anthocyanin is a major pigment in Asiatic hybrid lilies (Lilium spp.), with LhMYB12 being the first AN2 subgroup in monocots. In this study, the accumulation of pigments and gene transcripts during flower bud development was evaluated to determine the genes regulated by LhMYB12. LhMYB12 and anthocyanin biosynthesis genes showed the same transcription profiles, with LhMYB12 directly activating the promoters of chalcone synthase and dihydroflavonol 4-reductase. This indicates that LhMYB12 regulates both early and late genes, despite belonging to the AN2 subgroup. The cultivar Landini accumulated anthocyanin and flavonol. The contents of these pigments increased during the late stages of flower bud development; this might result from the coordinated expression of early and late genes. During the early stages of flower bud development, the tepals contained no flavonoids but accumulated cinnamic acid derivatives. These results indicate that the profiles of pigment accumulation and gene transcription in lily tepals are unique among angiosperm flowers.

Slantingly cross loading sample system enables simultaneous performance of separation and mixture to detect molecular interactions on thin-layer chromatography.

Anthocyanins are major flower pigments that can be affected by copigments, colorless compounds that can modify anthocyanin coloration to more intense and bluer. Thin-layer chromatography (TLC) is an available technique to separate and analyze anthocyanins and copigments. To easily and comprehensively detect copigments, we added function of mixture of compounds to TLC; by slantingly cross loading samples on TLC, compounds are symmetrically developed at various angle lines from the upper origin to individual R(f) values and cross each other in an orderly fashion, where mixture is simultaneously performed with separation. Occurrence of copigments can be detected as a coloration change on the developed line of anthocyanin. Pink sweet pea (Lathyrus odoratus L.) petals were analyzed by the cross-TLC and a more intense spot and a paler spot on the anthocyanin line were detected. As each spot overlapped with an ultraviolet absorbance line, each of these ultraviolet absorption compounds was purified and identified as kaempferol 3-rhamnoside and 2-cyanoethyl-isoxazolin-5-one, respectively. Whereas kaempferol 3-rhamnoside is a flavonoid and had a general copigment effect of more intense and bluer coloration change, 2-cyanoethyl-isoxazolin-5-one is a compound whose structure is outside of conventional categories of copigments and had a novel effect to change anthocyanin coloration paler while maintaining color tone. We determined that the search for copigments should be carried out without pre-existing prediction of structures and effects. We have shown that slantingly cross loading samples system on plate-type chromatography is an effective technique for such comprehensive analysis of molecular interaction.

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori.

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.

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.

Urakunoside, a new tetraglycosyl kaempferol from petals of the Wabisuke camellia cv. Tarokaja.

A new tetraglycosyl flavonol, 3-O-[2-O-xylosyl-6-O-(3-O-glucosyl-rhamnosyl) glucosyl] kaempferol was isolated from pale purplish-pink petals of Wabisuke camellia cv. Tarokaja with three known flavonols. It was named urakunoside after the species name of Tarokaja, Camellia uraku. Urakunoside was a major flavonol component in the Tarokaja petals, but was not detected in petals of Tarokaja's presumed ancestor species.

Isolation and characterization of the fragrant cyclamen O-methyltransferase involved in flower coloration.

Anthocyanin O-methyltransferase (OMT) is one of the key enzymes for anthocyanin modification and flower pigmentation. We previously bred a novel red-purple-flowered fragrant cyclamen (KMrp) from the purple-flowered fragrant cyclamen 'Kaori-no-mai' (KM) by ion-beam irradiation. Since the major anthocyanins in KMrp and KM petals were delphinidin 3,5-diglucoside and malvidin 3,5-diglucoside, respectively, inactivation of a methylation step in the anthocyanin biosynthetic pathway was indicated in KMrp. We isolated and compared OMT genes expressed in KM and KMrp petals. RT-PCR analysis revealed that CkmOMT2 was expressed in the petals of KM but not in KMrp. Three additional CkmOMTs with identical sequences were expressed in petals of both KM and KMrp. Genomic PCR analysis revealed that CkmOMT2 was not amplified from the KMrp genome, indicating that ion-beam irradiation caused a loss of the entire CkmOMT2 region in KMrp. In vitro enzyme assay demonstrated that CkmOMT2 catalyzes the 3' or 3',5' O-methylation of the B-ring of anthocyanin substrates. These results suggest that CkmOMT2 is functional for anthocyanin methylation, and defective expression of CkmOMT2 is responsible for changes in anthocyanin composition and flower coloration in KMrp.