出口 亜由美

KEY MESSAGE: Epidendrum produces 2n gametes with high frequency. This paper is the first to report on multiple pathways for forming 2n gametes, meiotic defeats, and pre-meiotic chromosome doubling. Unreduced 2n reproductive cells are predominantly involved in pathways that lead to polyploid plants. Although one of the most common pathways for inducing 2n gametes is through meiotic defects, a small set of isolated species alternatively generates 2n gametes from tetraploid pollen mother cells in the pre-meiotic phase. Hence, determining the mechanisms underlying 2n gamete formation is critical to improving breeding programmes and understanding plant evolution. We investigated sporads to reveal the pathway(s) accounting for the formation and frequencies of 2n gametes in wild species and interspecific hybrids in the genus Epidendrum. We investigated different types of sporads with varying frequencies, sizes, and viability in the wild species and hybrids of the genus Epidendrum. Large tetrad-estimated pre-meiotic chromosome doubling was observed in wild species. The Epidendrum is unique in that it forms 2n pollens via two pathways, namely, meiotic defects and pre-meiotic chromosome doubling. These two pathways of 2n pollen formation could influence the high diversity generation of polyploidy with different degrees of heterozygosity and genetic backgrounds in the genus Epidendrum. Therefore, these findings are proposed to influence polyploid breeding of Epidendrum via 2n pollen, helping us understand evolution and speciation via unreduced 2n gamete formation in Orchidaceae.

When used in closed-type plant factories, light-emitting diode (LED) illumination systems have the particular advantages of low heat emission and high luminous efficiency. The effects of illumination quality and intensity on the growth and morphogenesis of many plant species have been examined, but improvements are needed to optimize the illumination systems for better plant products with lower resource investments. In particular, new strategies are needed to reduce the wastage of plant products related to leaf senescence, and to better control the ingredients and appearance of leafy vegetables. Although the quality of light is often altered to change the characteristics of plant products, the transcriptional status underlying the physiological responses of plants to light has not been established. Herein, we performed a comprehensive gene expression analysis using RNA-sequencing to determine how red, blue, and red/blue LEDs and fluorescent light sources affect transcriptome involved in the leaf aging of leaf lettuce. The RNA-sequencing profiling revealed clear differences in the transcriptome between young and old leaves. Red LED light caused large variation between the two age classes, while a pure or mixed blue LED light spectrum induced fewer transcriptome differences between young and old leaves. Collectively, the expression levels of genes that showed homology with those of other model organisms provide a detailed physiological overview, incorporating such characteristics as the senescence, nutrient deficiency, and anthocyanin synthesis of the leaf lettuce plants. Our findings suggest that transcriptome profiles of leaf lettuce grown under different light sources provide helpful information to achieve better growth conditions for marketable and efficient green-vegetable production, with improved wastage control and efficient nutrient inputs.

<p>Flower color patterns are attractive traits of floricultural plants. However, the mechanisms underlying these traits remain mostly unknown. Carnation (Dianthus caryophyllus L.) and interspecific hybrids thereof exhibit many flower color patterns involving white margins on reddish petals, as observed in the cultivar 'Minerva'. Flowers with white margins also have white abaxial surfaces. We studied the factors regulating the formation of white margins and a white abaxial surface in flowers through analyses of pigments and related gene expression. HPLC analysis revealed an absence of anthocyanins in white margins, although the accumulation of other flavonoid pigments (flavonols) was almost identical between the dark-red central and white marginal regions of petals. RNA-seq analyses of the dark red central regions and white marginal regions of 'Minerva' petals resulted in the extraction of 18 genes related to anthocyanin biosynthesis and transportation, including some transcription factors, as candidate regulatory genes for the formation of white margins. Further analysis of the expression of these genes by real-time RT-PCR and a comparison of two white-margin-flowered cultivars and three red-unicolor-flowered cultivars indicated that the expressions of two bHLH transcription factor genes and seven structural genes were positively correlated with anthocyanin accumulation. Although DcbHLH1, which is a homolog of JAF13 in Petunia ×hybrida, was expressed in both flower color groups, DcbHLH2, a homolog of AN1, was expressed only in white-marginal-flowered cultivars. Moreover, in the petals of 'Minerva', the expression of those nine genes was repressed in the abaxial epidermal layer without red pigmentation conferred by anthocyanins. Therefore, we could postulate that the localized repression of both bHLH genes is involved in the formation of white margins in carnation petals by inducing the absence of anthocyanin synthesis and that the white abaxial surface of the petals may result from similar regulatory mechanisms. In particular, DcbHLH2 could act as a key gene because of its restricted expression only in cultivars with white-margined flowers.</p>

How genetic variations affect gene expression dynamics of field-grown plants remains unclear. Expression quantitative trait loci (eQTL) analysis is frequently used to find genomic regions underlying gene expression polymorphisms. This approach requires transcriptome data for the complete set of the QTL mapping population under the given conditions. Therefore, only a limited range of environmental conditions is covered by a conventional eQTL analysis. We sampled sparse time-series of field-grown rice from chromosome segment substitution lines (CSSLs) and conducted RNA-sequencing (RNA-Seq). Then, by using statistical analysis integrating meteorological data and the RNA-Seq data, we identified 1675 expression quantitative trait loci (eQTLs) leading to polymorphisms in expression dynamics under field conditions. A genomic region on chromosome 11 influences the expression of several defense-related genes in a time-of-day- and scaled-age-dependent manner. This includes the eQTLs that possibly influence the time-of-day- and scaled-age-dependent differences in the innate immunity between Koshihikari and Takanari. Based on the eQTL and meteorological data, we successfully predicted gene expression under environments different from training environments, and in rice cultivars with more complex genotypes than the CSSLs. Our novel approach of eQTL identification facilitated the understanding of the genetic architecture of expression dynamics under field conditions, which is difficult to assess by conventional eQTL studies. The prediction of expression based on eQTLs and environmental information would contribute to the understanding of plant traits under diverse field conditions. (224/250 words).

Chromosome numbers of wild species in the genus Epidendrum have previously been reported in the range of 2n= 24-240. However, the ploidy levels of cultivars in the given genus originating from interspecific hybridization among several wild species have never been reported. To elucidate their ploidy levels, we analyzed the chromosome number of cultivars and related wild species. Four wild species showed a new record of chromosome number in the present study, namely, E. radicans 2n= 38, 80, E. secundum 2n=60, and E. cinnabarinum 2n= 64. Six cultivars examined were revealed to have more chromosomes than the wild species, with a range of 2n= 84-164, suggesting that the cultivars have high polyploid levels. A significant correlation between the nuclear DNA amount and the chromosome number in wild species as well as cultivars was observed. Those cultivars originated from hybridization. Thus, the results from the present study suggest that the polyploid cultivars in this genus could have resulted from pollination involving unreduced gametes during breeding.

We examined the effects of five antimitotic agents using Antirrhinum majus L. 'Maryland True Pink' on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.

© 2020, The Society for In Vitro Biology. Dendrobium Stardust ‘Fire Bird’ is an interspecific hybrid between D. unicum and D. Ukon ‘Arai’ and is known to be sterile and prevents further breeding. Chromosome doubling is an essential method to increase the size of plant organs, such as flowers, and restore fertility of interspecific hybrids, which enables further breeding for ornamental traits. To induce chromosome doubling, the protocorm-like bodies (PLBs) of a given variety were treated in vitro for 6 to 48 h with 10 to 160 mg L−1 amiprofos-methyl (APM). Higher APM concentrations resulted in lower rates of survival of treated PLBs. Furthermore, subsequent growth of PLBs was hampered with prolonged APM treatment, although 10 and 20 mg L−1 APM was optimal for doubling chromosomes. Plantlets with a swollen and shorter stem were observed after the APM treatment and these plants were also chromosome-doubled and became a distinctive morphological marker for the primary selection of chromosome-doubled plants. Chromosome-doubled plants of D. Stardust ‘Fire Bird’ will be utilized for the future breeding of unique Dendrobium cultivars.

© 2020, Springer Nature B.V. Tetraploid plants of Antirrhinum majus L. were obtained from the seeds of the F1 variety ‘Maryland True Pink’ by amiprophos-methyl (APM) treatment and subsequent in vitro culture. Seeds were treated with 0, 10 and 20 mgl−1 APM for 1, 3, or 7 d (day) with or without osmopriming. Overall, the seedlings established from the seeds treated with 10 or 20 mgl−1 APM for 3–7 d exhibited malformation with rudimentary roots and swollen hypocotyls that eventually produced adventitious shoots. The highest adventitious shoot formation frequency reached 71.6% in non-primed seeds treated with 10 mgl−1 APM for 7 d; this decreased to 54.4% in osmoprimed seeds that received the same APM treatment. The plantlets from the adventitious shoots were identified as tetraploids at frequencies of 51.4% and 90.4%, respectively. Tetraploid frequency increased with higher concentration and/or longer duration of APM treatment and ranged from 0 to 65.9% in the non-primed seeds and 10.3% to 100% in the osmoprimed seeds. Osmopriming of seeds accelerated the induction of tetraploids by APM treatment at the same concentration and duration by 10.3–39.0%, except in the case of treatment with 20 mgl−1 APM for 3 d treatment. In the osmoprimed seeds treated with 20 mgl−1 APM for 7 d, all the plantlets from the adventitious shoots were revealed to be tetraploids. This study revealed that APM has dual effects, namely the inhibitory effect on the growth of seedlings and the stimulatory effect on the induction of adventitious shoots on the hypocotyl of seedlings.

RNA-Seq is a powerful method for transcriptome analysis used in varied field of biology. Although several commercial products and hand-made protocols enable us to prepare RNA-Seq library from total RNA, their cost are still expensive. Here, we established a low-cost and multiplexable whole mRNA-Seq library preparation method for illumine sequencers. In order to reduce cost, we used cost-effective and robust commercial regents with small reaction volumes. This method is a whole mRNA-Seq, which can be applied even to non-model organisms lacking the transcriptome references. In addition, we designed large number of 3' PCR primer including 8 nucleotides barcode sequences for multiplexing up to three hundreds samples. To summarize, it is possible with this protocol to prepare 96 directional RNA-Seq libraries from purified total RNA in three days and can be pooled for up to three hundred libraries. This is beneficial for large scale transcriptome analysis in many fields of animals and plant biology.

A cultivar of Catharanthus roseus, 'Jams 'N Jellies Blackberry', was investigated for its rare blackish flower color by comparing several factors affecting flower color with another ten C. roseus cultivars that had red to purple flower colors. Petals of 'Jams 'N Jellies Blackberry' accumulated only one novel anthocyanin, peonidin 3-robinobioside (Pn 3-rob), whereas those of all the other cultivars contained multiple anthocyanins. Moreover, although 'Jams 'N Jellies Blackberry' accumulated only one anthocyanin, Pn 3-rob, the total anthocyanin concentration was 2.5-fold higher in 'Jams 'N Jellies Blackberry' than in the cultivar with the second-highest anthocyanin concentration 'Equator Deep Apricot', which had orange red flowers. Microscopic observation of transverse sections of petals revealed that only 'Jams 'N Jellies Blackberry' exhibited aggregation of anthocyanins, namely anthocyanic vacuolar inclusions (AVIs), in the vacuoles of petal epidermal cells. Furthermore, the pH of petals of 'Jams 'N Jellies Blackberry' was approximately 6.0, which was significantly higher than that of all the other cultivars examined, which were in the range 5.0-5.6. Using purified Pn 3-rob and three other anthocyanins, 7-methylcyanidin 3-robinobioside, rosinidin 3-robinobioside, and hirstidin 3-robinobioside, which were the main anthocyanins accumulated in cultivars other than 'Jams 'N Jellies Blackberry', the color of their solutions was measured in vitro at pH values of 3.0, 5.0, and 6.0. Pn 3-rob did not exhibit the darkest color (lowest C-star and L-star) at all pH conditions investigated. However, at pH 5.0 and 6.0, Pn 3-rob recrystallized immediately after dissolution, and the solution retained the dark color even though slight discoloration occurred. The feature of aggregation of anthocyanins in the solution was almost identical to that in the AVIs observed in petal epidermal cells of 'Jams 'N Jellies Blackberry'. The color of the anthocyanin solution was darker at pH 6.0 than at pH 5.0 for all types of anthocyanin tested. Based on these results, we could postulate that multiple factors, such as the type and high concentration of anthocyanins, high pH of the petals, and formation of AVIs, could be involved in the development of the rare blackish flower color of 'Jams 'N Jellies Blackberry'. Further studies should be conducted to reveal the degree of contribution of each factor to blackish flower color using progenies of 'Jams 'N Jellies Blackberry' with various phenotypes and genotypes.

Recently, increasing attempts have been made to understand how plant genes function in natura. In this context, transcriptional profiles represent plant physiological status in response to environmental stimuli. Herein, we combined high-throughput RNA-Seq with insect survey data on 19 accessions of Arabidopsis thaliana grown at a field site in Switzerland. We found that genes with the gene ontology (GO) annotations of "glucosinolate biosynthetic process" and "response to insects" were most significantly enriched, and the expression of these genes was highly variable among plant accessions. Nearly half of the total expression variation in the glucosinolate biosynthetic genes (AOPs, ESM1 , ESP, and TGG1) was explained by among-accession variation. Of these genes, the expression level of AOP3 differed among Col-0 accession individuals depending on the abundance of the mustard aphid (Lipaphis erysimi). We also found that the expression of the major cis-jasmone activated gene CYP81D11 was positively correlated with the number of flea beetles (Phyllotreta striolata and Phyllotreta atra). Combined with the field RNA-Seq data, bioassays confirmed that AOP3 was up-regulated in response to attack by mustard aphids. The combined results from RNA-Seq and our ecological survey illustrate the feasibility of using field transcriptomics to detect an inducible defense, providing a first step towards an in natura understanding of biotic interactions involving phenotypic plasticity.

Plants sense and respond to light via multiple photoreceptors including phytochrome. The decreased ratio of red to far-red light that occurs under a canopy triggers shade-avoidance responses, which allow plants to compete with neighboring plants. The leaf acts as a photoperceptive organ in this response. In this study, we investigated how the shade stimulus is spatially processed within the cotyledon. We performed transcriptome analysis on microtissue samples collected from vascular and nonvascular regions of Arabidopsis (Arabidopsis thaliana) cotyledons. In addition, we mechanically isolated and analyzed the vascular tissue. More genes were up-regulated by the shade stimulus in vascular tissues than in mesophyll and epidermal tissues. The genes up-regulated in the vasculature were functionally divergent and included many auxin-responsive genes, suggesting that various physiological/developmental processes might be controlled by shade stimulus in the vasculature. We then investigated the spatial regulation of these genes in the vascular tissues. A small vascular region within a cotyledon was irradiated with far-red light, and the response was compared with that when the whole seedling was irradiated with far-red light. Most of the auxin-responsive genes were not fully induced by the local irradiation, suggesting that perception of the shade stimulus requires that a wider area be exposed to far-red light or that a certain position in the mesophyll and epidermis of the cotyledon be irradiated. This result was consistent with a previous report that auxin synthesis genes are up-regulated in the periphery of the cotyledon. Hence, auxin acts as an important intraorgan signaling factor that controls the vascular shade response within the cotyledon.

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 onlymonochromatic-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.

In this study, we revealed how the petals of Saintpaulia fuse into a corolla by using pinwheel phenotype cultivars. Striped patterns in petal, called pinwheel in Saintpaulia, are attractive phenotypes and thought to be the result of periclinal chimerism. For the selection of a genuine periclinal chimeric cultivar from three pinwheel cultivars, adventitious shoots were induced from leaf lamina. Shoot regeneration was observed from the epidermis in all cultivars by microscopic observation. All regenerated shoots from 'Kaname' flowered as monochromatic pink flowers, corresponding to an Ll phenotype of the cultivar. From the other two cultivars, many shoots flowered not only as an epidermal phenotype but also as a phenotype of the inner layer. In addition, shoot regeneration was induced from epidermis-peeled petioles from these three cultivars. All shoots from 'kaname' flowered as monochromatic blue flowers, corresponding to an L2 phenotype. On the other hand, many shoots from 'Kilauea' flowered not only as monochromatic flowers, corresponding to an L2 phenotype, but also as bi-colored flowers. 'Innocent Pink' did not produce shoots from epidermal-peeled petioles. These results suggested that 'Kaname' is a genuine periclinal chimera, while the other two cultivars have other mechanisms for pinwheel expression. Genomic PCR using primers that amplifies almost the full length of flavonoid 3',5'-hydroxylase (F3'5'H) revealed the gene to be non-functional in pink flowers from Ll of 'Kaname'. From monochromatic pink plants and pink portions of the corolla of 'Raname', full-length F3'5'H was not amplified. Similar results were obtained by quantitative PCR. Finally, we observed the fused portion of the petals and revealed that the petal fusion did not occur by postgenital fusion but by "connection". The process, in Saintpaulia, comprises periclinal cell division in Ll during petal development, active cell division at the edge of the petal, adhesion to the next petals, and fusion. These steps create a striped flower color in Saintpaulia.

The black flower color of dahlias (Dahlia variabilis) has been suggested to be attributed to a high accumulation of cyanidin (Cy)-based anthocyanins. A possible explanation for this effect is that Cy-based anthocyanins in dahlias contribute more to the black flower color than pelargonidin (Pg)-based anthocyanins by lowering petal lightness (L*) and chroma (C*), but no obvious evidence has been reported. In this study, four major anthocyanins accumulated in dahlia petals, 3,5-diglucoside (3,5diG) and 3-(6 ''-malonylglucoside)-5-glucoside (3MG5G) of Pg and Cy, were purified and their colors were evaluated in vitro at various pHs (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, or 7.0) and various concentrations (0.25, 0.5, 1.0, 2.0, or 3.0 mg.mL(-1) at pH 5.0 or pH 3.0). The color of solution of purified anthocyanins varied depending on pH. At pH 5.0, which is approximately the same as pH of dahlia petals, and at pH 3.0, at which anthocyanins are relatively stable, the L* and C* of Cy 3,5diG were similar to or higher than those of Pg 3,5diG, suggesting that Cy 3,5diG did not contribute more to the black flower coloring than Pg 3,5diG. On the other hand, the L* and C* of Cy 3MG5G were significantly lower than those of Pg 3MG5G, particularly above 2.0 mg.mL(-1), suggesting that Cy 3MG5G contributed more than Pg 3MG5G. A similar tendency was observed in the color measurement of mixed anthocyanins in various proportion of Pg and Cy. The L* and C* of Pg 3MG5G were much higher than those of the other three anthocyanins; therefore, its color was considered to be the farthest from black among the four anthocyanins. The accumulated amount of 3MG5G-type anthocyanins was much higher than that of 3,5diG-type anthocyanins in all nine cultivars, although the proportion of Pg- and Cy-based anthocyanins varied among the cultivars. Considering these results, it was suggested that because 3MG5G-type anthocyanins predominantly accumulate in petals, and Cy 3MG5G has a significantly higher contribution to lowering L* and C* than Pg 3MG5G, the high accumulation of Cy-based anthocyanins is critical for the black flower coloring of dahlias. The contribution of each anthocyanin is considered to depend on the structure; therefore, identifying the anthocyanin with the highest contribution to lowering L* and C* may enable the production of black flowers in various species through the high accumulation of the anthocyanin in petals.

セントポーリア'Tomorrow's Snowy Night'の親個体および組織培養変異体として出現した黄色変異体の花弁から黄色色素を単離し，化学分析およびスペクトル分析によりchalcononaringenin 2´,4´-diglucosideであることを明らかにした．黄色変異体では主に蓄積部位の拡大により花弁全体でのchalcononaringenin 2´,4´-diglucoside蓄積量が増加していた．また，chalcononaringenin 2´,4´-diglucosideはセントポーリアの花糸においては花弁での蓄積とは関係なく蓄積しており，この合成能力は黄色品種以外にも広く存在すると考えられた．これらの情報はセントポーリアにおけるchalcononaringenin 2´,4´-diglucoside合成の遺伝子機構の解明に役立つだろう．

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.

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.

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.

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.