Yasumasa Morita

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.

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.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

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.

A petal-specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops.

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal-specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal-specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal-specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β-glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal-specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA-like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal-specific promoter in molecular breeding of floricultural crops.

An Active CACTA-Family Transposable Element is Responsible for Flower Variegation in Wild Soybean Glycine soja

A plant producing flowers with purple and white variegation was discovered in an accession of Glycine soja Siebold & Zucc. that was introduced from Russia. The mutant line was designated as B00146‐m. Lines with white flowers (B00146‐w) and purple flowers (B00146‐r) were developed from the progeny of B00146‐m. The flower color was controlled by the W1 locus encoding a flavonoid 3′5′‐hydroxylase (F3′5′H). The allele for variegated flowers was designated as w1‐m. The gene symbol was approved by the Soybean Genetics Committee. Polymerase chain reaction (PCR) suggested that a DNA fragment with a molecular size of ∼3.9 kb was inserted in the first exon of the F3′5′H gene in B00146‐m whereas such insertion was not observed in B00146‐w and B00146‐r. These results suggested that an active mobile element was inserted in the first exon and was responsible for flower variegation. The inserted fragment was identified as a 3883 bp long CACTA‐family transposable element and it was designated as Tgs1. Similarity of overall sequence and terminal inverted repeats suggested that Tgs1 and the soybean lectin gene transposable element Tgm1 make up a subgroup. Frequency of germinal reversion was low probably due to the integration into an exon. Tgs1 had a truncated version of the transposase gene and may be a nonautonomous element.

An acylated pelargonidin 3-sophoroside from the pale-brownish red flowers of Ipomoea nil

A structurally new acylated anthocyanin was isolated from the pale-brownish red flowers of a duskish-2 mutant in the Japanese morning glory (Ipomoea nil or Pharbitis nil) as a major pigment together with a known anthocyanin. The new pigment was determined as pelargonidin 3-O-[2-O-(6-O-(trans-3-O-(β-glucopyranosyl)caffeoyl)-β-glucopyranosyl)-β-glucopyranoside], and the known anthocyanin was identified to be pelargonidin 3-sophoroside.

Recent advances in flower color variation and patterning of Japanese morning glory and petunia

The Japanese morning glory (Ipomoea nil) and petunia (Petunia hybrida), locally called “Asagao” and “Tsukubane-asagao”, respectively, are popular garden plants. They have been utilized as model plants for studying the genetic basis of floricultural traits, especially anthocyanin pigmentation in flower petals. In their long history of genetic studies, many mutations affecting flower pigmentation have been characterized, and both structural and regulatory genes for the anthocyanin biosynthesis pathway have been identified. In this review, we will summarize recent advances in the understanding of flower pigmentation in the two species with respect to flower hue and color patterning. Regarding flower hue, we will describe a novel enhancer of flavonoid production that controls the intensity of flower pigmentation, new aspects related to a flavonoid glucosyltransferase that has been known for a long time, and the regulatory mechanisms of vacuolar pH being a key determinant of red and blue coloration. On color patterning, we describe particular flower patterns regulated by epigenetic and RNA-silencing mechanisms. As high-quality whole genome sequences of the Japanese morning glory and petunia wild parents (P. axillaris and P. inflata, respectively) were published in 2016, further study on flower pigmentation will be accelerated.