A novel aldo-keto reductase gene is involved in 6'-deoxychalcone biosynthesis in dahlia (Dahlia variabilis).

MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

Anthocyanin regulatory and structural genes associated with violet flower color of Matthiola incana.

MAIN CONCLUSION: MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana. Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in 'Vintage Lavender' increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3'H, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of 'Vintage Lavender'. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than 'Vintage Lavender'. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3'H, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1-MibHLH2-MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

Post-transcriptional silencing of chalcone synthase is involved in phenotypic lability in petals and leaves of bicolor dahlia (Dahlia variabilis) 'Yuino'.

MAIN CONCLUSION: Post-transcriptional gene silencing (PTGS) of a chalcone synthase ( DvCHS2 ) occurred in the white part of bicolor petals and flavonoid-poor leaves; however, it did not in red petals and flavonoid-rich leaves. Petal color lability is a prominent feature of bicolor dahlia cultivars, and causes plants to produce not only original bicolor petals with colored bases and pure white tips, but also frequently single-colored petals without white tips. In this study, we analysed the molecular mechanisms that are associated with petal color lability using the red-white bicolor cultivar 'Yuino'. Red single-colored petals lose their white tips as a result of recover of flavonoid biosynthesis. Among flavonoid biosynthetic genes including four chalcone synthase (CHS)-like genes (DvCHS1, DvCHS2, DvCHS3, and DvCHS4), DvCHS1 and DvCHS2 had significantly lower expression levels in the white part of bicolor petals than in red petals, while DvCHS3, DvCHS4, and other flavonoid biosynthetic genes had almost the same expression levels. Small RNAs from the white part of a bicolor petal were mapped onto DvCHS1 and DvCHS2, while small RNAs from a red single-colored petal were not mapped onto any of the four CHS genes. A relationship between petal color and leaf flavonoid accumulation has previously been demonstrated, whereby red petal-producing plants accumulate flavonoids in their leaves, while bicolor petal-producing plants tend not to. The expression level of DvCHS2 was down-regulated in flavonoid-poor leaves and small RNAs from flavonoid-poor leaves were mapped onto DvCHS2, suggesting that the down-regulation of DvCHS2 in flavonoid-poor leaves occurs post-transcriptionally. Genomic analysis also suggested that DvCHS2 is the key gene involved in bicolor formation. Together, these results suggest that post-transcriptional gene silencing of DvCHS2 plays a key role in phenotypic lability in this bicolor dahlia.

Tobacco streak virus (strain dahlia) suppresses post-transcriptional gene silencing of flavone synthase II in black dahlia cultivars and causes a drastic flower color change.

MAIN CONCLUSION: Tobacco streak virus suppressed post-transcriptional gene silencing and caused a flower color change in black dahlias, which supported the role of cyanidin-based anthocyanins for black flower appearance. Black flower color of dahlia (Dahlia variabilis) has been attributed, in part, to the high accumulation of cyanidin-based anthocyanins that occurs when flavone synthesis is reduced because of post-transcriptional gene silencing (PTGS) of flavone synthase II (DvFNS). There are also purple-flowering plants that have emerged from a black cultivar 'Kokucho'. We report that the purple color is not caused by a mutation, as previously thought, but by infection with tobacco streak virus (TSVdahlia), which suppresses the PTGS of DvFNS. When TSVdahlia was eliminated from the purple-flowering 'Kokucho' by leaf primordia-free shoot apical meristem culture, the resulting flowers were black. TSVdahlia-infected purple flowers had lower numbers of siRNAs to DvFNS than black flowers, suggesting that TSVdahlia has a silencing suppressor. The graft inoculation of other black cultivars with TSVdahlia altered their flower color drastically except for 'Fidalgo Blacky', a very deep black cultivar with the highest amount of cyanidin-based anthocyanins. The flowers of all six TSVdahlia-infected cultivars accumulated increased amounts of flavones and reduced amounts of cyanidin-based anthocyanins. 'Fidalgo Blacky' remained black despite the change in pigment accumulation, and the amounts of cyanidin-based anthocyanins in its TSVdahlia-infected plants were still higher than those of other cultivars. We propose that black flower color in dahlia is controlled by two different mechanisms that increase the amount of cyanidin-based anthocyanins: DvFNS PTGS-dependent and -independent mechanisms. If both mechanisms occur simultaneously, the flower color will be blacker than if only a single mechanism is active.

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars.

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars.

The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity.

Simultaneous post-transcriptional gene silencing of two different chalcone synthase genes resulting in pure white flowers in the octoploid dahlia.

Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis).

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Tissue culture-induced flower-color changes in Saintpaulia caused by excision of the transposon inserted in the flavonoid 3', 5' hydroxylase (F3'5'H) promoter.

The variegated Saintpaulia cultivar Thamires (Saintpaulia sp.), which has pink petals with blue splotches, is generally maintained by leaf cuttings. In contrast, tissue culture-derived progeny of the cultivar showed not only a high percentage of mutants with solid-blue petals but also other solid-color variants, which have not been observed from leaf cuttings. Solid-color phenotypes were inherited stably by their progeny from tissue culture. Petals from each solid-color variant were analyzed by high-performance liquid chromatography and shown to contain different proportions of three main anthocyanin derivatives: malvidin, peonidin, and pelargonidin. Analysis of flavonoid 3', 5'-hydroxylase (F3'5'H) sequences showed no differences in the coding region among the variants and variegated individuals. However, a transposon belonging to the hAT superfamily was found in the promoter region of variegated individuals, and the presence of transposon-related insertions or deletions correlated with the observed flower-color phenotypes. Solid-blue flower mutants contained 8-base pair (bp) insertions (transposon excision footprints), while solid-pink mutants had 58- to 70-bp insertions, and purple- and deep-purple mutants had 21- and 24-bp deletions, respectively. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed that F3'5'H expression levels correlated with insertions and deletions (indels) caused by hAT excision, resulting in flower-color differences. Our results showed that tissue culture of Saintpaulia 'Thamires' elicits transposon excision, which in turn alters F3'5'H expression levels and flower colors.

Flavonoids in the flowers of Primula ×polyantha Mill. and Primula primulina (Spreng.) H. Hara (Primulaceae).

Two undescribed anthocyanins and two undescribed flavonols were isolated from the flowers of Primula ×polyantha Mill., along with five known anthocyanins and four known flavonols. The two undescribed anthocyanins and the two undescribed flavonols were determined to be hirsutidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside, 7-O-methyl-petunidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside, quercetin 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside], and kaempferol 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside] using chemical and spectroscopic methods. They were also found in the flowers of the Himalayan wild species, Primula primulina (Spreng.) H. Hara except for quercetin 3-O-ß-[(6""-acetylglucopyranosyl)-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside]. The flower color variations of P. ×polyantha cultivars, reflected by the hue values (b*/a*) of the colors, were due to the glycosidic patterns in the anthocyanins and their concentrations in the petals. Moreover, in the P. ×polyantha cultivars with violet-blue flowers, both the intermolecular copigmentation occurs between hirsutidin 3-O-ß-galactopyranoside-5-O-ß-glucopyranoside and another flavonol, quercetin 3-O-ß-glucopyranosyl-(1 â†’ 2)-ß-glucopyranosyl-(1 â†’ 6)-ß-glucopyranoside. Moreover, the flower color variation was affected by the pH value.

Acylated pelargonidin glycosides from the red-purple flowers of Iberis umbellata L. and the red flowers of Erysimum × cheiri (L.) Crantz (Brassicaceae).

Five previously undescribed acylated pelargonidin 3-sophoroside-5-glucosides (pigments 2-6) were isolated from the red-purple flowers of Iberis umbellata L. 'Candycane Rose' and 'Candycane Red', in addition to a known one (pigment 1). The structures of five undescribed acylated anthocyanins were determined by chemical and spectroscopic methods to be pelargonidin 3-O-[2-O-(2-O-("acyl-A")-ß-glucopyranosyl)-6-O-("acyl-B")-ß-glucopyranoside]-5-O-[6-O-(malonyl)-ß-glucopyranoside], in which the "acyl-A" group was either trans-sinapic (2), trans-ferulic (3), trans-sinapic (4), trans-ferulic (5), or trans-ferulic acid (6), and "acyl-B" was either glucosyl-trans-p-coumaric acid (2), glucosyl-trans-p-coumaric acid (3), trans-feruloyl-glucosyl-trans-p-coumaric acid (4), trans-feruloyl-glucosyl-trans-p-coumaric acid (5), or glucosyl-trans-feruloyl-glucosyl-trans-p-coumaric acid (6). Moreover, three previously undescribed acylated pelargonidin 3-sambubioside-5-glucosides (pigments 7, 8, and 10) and one undescribed acylated pelargonidin 3-(3X-glucosylsambubioside)-5-glucoside (pigment 9) were isolated from the red flowers of Erysimum × cheiri (L.) Crantz 'Aurora' as major anthocyanins. The structures of the three undescribed acylated pelargonidin 3-sambubioside-5-glucosides were determined to be pelargonidin 3-O-[2-O-(2-O-("acyl-C")-ß-xylopyranosyl)-6-O-("acyl-D")-ß-glucopyranoside]-5-O-(ß-glucopyranoside), in which the "acyl-C" group was either non (7), non (8), or trans-p-coumaric acid (10) and "acyl-D" was either trans-p-coumaric (7), trans-ferulic (8), or trans-p-coumaric acid (10). Moreover, a previously undescribed acylated pelargonidin 3-(3X-glucosylsambubioside)-5-glucoside was identified to be pelargonidin 3-O-[2-O-(2-O-(trans-p-coumaroyl)-3-O-(ß-glucopyranosyl)-ß-xylopyranosyl)-6-O-(trans-p-coumaroyl)-ß-glucopyranoside]-5-O-(ß-glucopyranoside) (9). In addition, the distribution of anthocyanidins structural elements in 24 Brassicaceous species is compared.

7-O-Methylated anthocyanidin glycosides from Catharanthus roseus.

Anthocyanins were isolated from orange-red flowers of Catharanthus roseus cv 'Equator Deep Apricot', and identified as rosinidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] (1), and also 7-O-methylcyanidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] (2) by chemical and spectroscopic methods. Pigment 1 was found to be a major anthocyanin in the flowers of this cultivar. By contrast, the distribution of rosinidin glycosides is very limited in plants, and reported only in the flowers of Primula. Pigment 2 was found in smaller concentrations, but its aglycone, 7-O-methylcyanidin, has been reported only once before, from the fruit of mango.

Triacylated cyanidin 3-(3X-glucosylsambubioside)-5-glucosides from the flowers of Malcolmia maritima.

Three acylated cyanidin 3-(3(X)-glucosylsambubioside)-5-glucosides (1-3) and one non-acylated cyanidin 3-(3(X)-glucosylsambubioside)-5-glucoside (4) were isolated from the purple-violet or violet flowers and purple stems of Malcolmia maritima (L.) R. Br (the Cruciferae), and their structures were determined by chemical and spectroscopic methods. In the flowers of this plant, pigment 1 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-beta-D-glucopyranoside]-5-O-[6-O-(malonyl)-(beta-D-glucopyranoside) as a major pigment, and a minor pigment 2 was determined to be the cis-p-coumaroyl isomer of pigment 1. In the stems, pigment 3 was determined to be cyanidin 3-O-[2-O-(2-O-(trans-sinapoyl)-3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-6-O-(trans-p-coumaroyl)-beta-d-glucopyranoside]-5-O-(beta-D-glucopyranoside) as a major anthocyanin, and also a non-acylated anthocyanin, cyanidin 3-O-[2-O-(3-O-(beta-D-glucopyranosyl)-beta-D-xylopyranosyl)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside) was determined to be a minor pigment (pigment 4). In this study, it was established that the acylation-enzymes of malonic acid has important roles for the acylation of 5-glucose residues of these anthocyanins in the flower-tissues of M. maritima; however, the similar enzymatic reactions seemed to be inhibited or lacking in the stem-tissues.

Tetra-acylated cyanidin 3-sophoroside-5-glucosides from the flowers of Iberis umbellata L. (Cruciferae).

The structures of 11 acylated cyanidin 3-sophoroside-5-glucosides (pigments 1-11), isolated from the flowers of Iberis umbellata cultivars (Cruciferae), were elucidated by chemical and spectroscopic methods. Pigments 1-11 were acylated with malonic acid, p-coumaric acid, ferulic acid, sinapic acid and/or glucosylhydroxycinnamic acids. Pigments 1-11 were classified into four groups by the substitution patterns of the linear acylated residues at the 3-position of the cyanidin. In the first group, pigments 1-3 were determined to be cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which the acyl moiety varied with none for pigment 1, ferulic acid for pigment 2 and sinapic acid for pigment 3. In the second one, pigments 4-6 were cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(4-O-(beta-glucopyranosyl)-trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which the acyl moiety varied with none for pigment 4, ferulic acid for pigment 5 and sinapic acid for pigment 6. In the third one, pigments 7-9 were cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(4-O-(6-O-(trans-feruloyl)-beta-glucopyranosyl)-trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which the acyl moiety varied with none for pigment 7, ferulic acid for pigment 8, and sinapic acid for pigment 9. In the last one, pigments 10 and 11 were cyanidin 3-O-[2-O-(2-O-(acyl)-beta-glucopyranosyl)-6-O-(4-O-(6-O-(4-O-(beta-glucopyranosyl)-trans-feruloyl)-beta-glucopyranosyl)-trans-p-coumaroyl)-beta-glucopyranoside]-5-O-[6-O-(malonyl)-beta-glucopyranoside], in which acyl moieties were none for pigment 10 and ferulic acid for pigment 11. The distribution of these pigments was examined in the flowers of four cultivars of I. umbellata by HPLC analysis. Pigment 1 acylated with one molecule of p-coumaric acid was dominantly observed in purple-violet cultivars. On the other hand, pigments (9 and 11) acylated with three molecules of hydroxycinnamic acids were observed in lilac (purple-violet) cultivars as major anthocyanins. The bluing effect and stability on these anthocyanin colors were discussed in relation to the molecular number of hydroxycinnamic acids in these anthocyanin molecules.

7-Polyacylated delphinidin 3,7-diglucosides from the blue flowers of Leschenaultia cv. Violet Lena.

The triacyl anthocyanins, Leschenaultia blue anthocyanins 1 and 2 (LBAs 1 and 2) were isolated from the blue flowers of Leschenaultia R. Br. cv. Violet Lena (Goodeniaceae), in which LBA 1 was present as a dominant pigment. The structure of LBA 1 was elucidated to be delphinidin 3-O-[6-O-(malonyl)-beta-D-glucopyranoside]-7-O-[6-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranoside] by application of chemical and spectroscopic methods. Since LAB 2 was isolated in small amount, its structure was tentatively assigned as either delphinidin 3-(malonylglucoside)-7-[(glucosyl-p-coumaroyl)-(glucosylcaffeoyl)-glucoside] or delphinidin 3-(malonyl-glucoside)-7-[(glucosyl-caffeoyl)(glucosyl-p-coumaroyl)-glucoside]. This is the first report of the occurrence of 7-polyacylated anthocyanins in the family of Goodeniaceae, although others have been found in the families of the Ranunculaceae, Campanulaceae, and Compositae. Moreover, delphinidin 3-glycoside-7-di-(glucosylcaffeoyl)-glucoside has been reported only in the flowers of Platycodon grandiflorum (Campanulaceae). From a chemotaxonomical viewpoint, the Goodeniaceae may be closely related to the Campanulaceae.

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.

Histogen Layers Contributing to Adventitious Bud Formation Are Determined by their Cell Division Activities.

Saintpaulia ionantha is propagated by adventitious buds in horticulture, and periclinal chimeral cultivars are usually difficult to propagate. However, some periclinal chimeral cultivars can be propagated with adventitious buds, and the mechanism of which has been unknown. Striped flower cultivars "Kaname," "Concord," and "Monique" were used to investigate what causes flower color separation in adventitious shoot-derived plants by tissue culture. These cultivars were revealed to have mutated flavonoid 3', 5' hydroxylase (SiF3'5'H), WDR1 (SiWDR1), or flavonoid 3 hydroxylase (SiF3H), respectively, in their L1 layer. From our previous study using "Kaname," all flowers from adventitious shoots were colored pink, which was the epidermal color of mother plants' flowers. We used "Concrd" and "Monique" from which we obtained not only monochromatic-colored plants the same as the epidermal color of mother plants, but also plants with a monochromatic colored plants, same as the subepidermal color, and a striped flower color the same as mother plants. Histological observations revealed that epidermal cells divided actively at 14 d after culture and they were involved in the formation of adventitious shoots in the cultured leaf segments of "Kaname." On the other hand, in "Concord" and "Monique," the number of divided cells in the subepidermis was rather higher than that of epidermal cells, and subepidermal cells were sometimes involved in shoot formation. In addition, the plant and leaf size of L1-derived plants from "Concord" and "Monique" were non-vigorous and smaller than those derived from the subepidermal layer. In conclusion, periclinal chimeral cultivars of Saintpaulia can be divided into two types. One type has a high cell division activity in the L1 layer, from which only single flower-colored plants derived from L1 can be obtained as adventitious shoots. Another type has a low cell division activity in the L1 layer, from which striped flower-colored plants the same as mother plants derived from several layers including L1 can be obtained as adventitious shoots. In the periclinal chimeral cultivar capable of propagation with adventitious shoots, the possibility was shown that cells in the L2 layer could form shoots by involving cells of the L1 layer with a low division activity.

Acylated cyanidin 3-sambubioside-5-glucosides in three garden plants of the Cruciferae.

Seven acylated cyanidin 3-sambubioside-5-glucosides were isolated from the flowers of three garden plants in the Cruciferae. Specifically, four pigments were isolated from Lobularia maritima (L.) Desv., together with a known pigment, as well as, three pigments from Lunaria annua L., and two known pigments from Cheiranthus cheiri L. These pigments were determined to be cyanidin 3-O-[2-O-((acyl-II)-(beta-d-xylopyranosyl))-6-O-(acyl-I)-beta-d-glucopyranoside]-5-O-[6-O-(acyl-III)-beta-d-glucopyranoside], in which the acyl-I group is represented by glucosyl-p-coumaric acid, p-coumaric acid and ferulic acid, acyl-II by caffeic acid and ferulic acid, and acyl-III by malonic acid, respectively. The distribution and biosynthesis of acylated cyanidin 3-sambubioside-5-glucosides are discussed according to the variations of acylation and glucosylation at their 3-sambubiose residues.

Acylated anthocyanins from the violet-blue flowers of Orychophragonus violaceus.

Three acylated cyanidin 3-sambubioside-5-glucosides (1-3) were isolated from the violet-blue flowers of Orychophragonus violaceus, and their structures were determined by chemical and spectroscopic methods. Two of those acylated anthocyanins (1 and 3) were cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-acyl)-beta-D-glucopyranoside]-5-O-(6-O-malonyl-beta-D-glucopyranoside)s, in which the acyl groups were p-coumaric acid for 1, and sinapic acid for 3, respectively. The last anthocyanin 2 was cyanidin 3-O-[2-O-(2-O-(4-O-(6-O-(4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-glucopyranosyl)-trans-caffeoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-trans-feruloyl)-beta-D-glucopyranoside]-5-O-beta-D-glucopyranoside. In these flowers, the anthocyanins 2 and 3 were present as dominant pigments, and 1 was obtained in rather small amounts.

Identification of the glucosyltransferase that mediates direct flavone C-glucosylation in Gentiana triflora.

The major flavonoids accumulated in leaves of Japanese gentian (Gentiana triflora) were determined as isoorientin (luteolin 6-C-glucoside) and isoorientin 4'-glucoside. A cDNA (GtUF6CGT1) was isolated that encoded the UDP-glucose-dependent glucosyltransferase that is involved in C-glycosylflavone biosynthesis. The recombinant GtUF6CGT1 protein could transfer a glucose group to the C6 position of a flavone skeleton through C-linkage, using UDP-glucose as the glucosyl donor. These C-glycosylflavones also accumulated in petals. A good correlation was observed between GtUF6CGT1 expression and C-glycosylflavone accumulation in leaves and petals. GtUF6CGT1 is the first reported C-glucosyltransferase that mediates direct C-glucosylation of the flavone skeleton.