Petal Color Research Articles

Genetic delimitation of Oreocharis species from Hainan Island

Hainan Island harbours an extraordinary diversity of Gesneriaceae with 14 genera and 23 species, amongst which two species and one variety are recognised in the genus Oreocharis. These three Oreocharis taxa are all Hainan-endemics and show a complex geographical distribution pattern with considerable morphological intermixtures. In this study, we combined DNA (nuclear ITS sequences and cpDNAtrnL-trnF and ycf1b) to evaluate genetic delimitation for 12 Oreocharis populations from the island, together with morphological similarity analysis using 16 morphological traits. The results showed Hainan Oreocharis taxa were monophyletic with relative low genetic diversity within populations, highly significant genetic differentiation amongst populations and a significant phylogeographical structure. The 12 populations formed three genetically distinct groups, roughly correspondent to the currently recognised two species and one unknown lineage. The PCA analyses of morphological traits indicate three distinctive groups, differing mainly in petal colour and corolla shapes. The roles of river and mountain isolations in the origin and distribution of these three lineages are discussed.

Genetic delimitation of Oreocharis species from Hainan Island

Hainan Island harbours an extraordinary diversity of Gesneriaceae with 14 genera and 23 species, amongst which two species and one variety are recognised in the genusOreocharis. These threeOreocharistaxa are all Hainan-endemics and show a complex geographical distribution pattern with considerable morphological intermixtures. In this study, we combined DNA (nuclear ITS sequences and cpDNAtrnL-trnF andycf1b) to evaluate genetic delimitation for 12Oreocharispopulations from the island, together with morphological similarity analysis using 16 morphological traits. The results showed HainanOreocharistaxa were monophyletic with relative low genetic diversity within populations, highly significant genetic differentiation amongst populations and a significant phylogeographical structure. The 12 populations formed three genetically distinct groups, roughly correspondent to the currently recognised two species and one unknown lineage. The PCA analyses of morphological traits indicate three distinctive groups, differing mainly in petal colour and corolla shapes. The roles of river and mountain isolations in the origin and distribution of these three lineages are discussed.

Genetic engineering as a way to obtain ornamental plants with a changed flower color

An important trend in the field of floriculture is the creation of new varieties of ornamental plants, among which varieties with unusual color are most in demand. To this end, traditional breeding and selection programs have been successfully applied for many years. However, currently genetic engineering is able to offer an alternative way to obtain new forms and varieties. Anthocyanins belonging to flavonoids, betalains and carotenoids are the main types of pigments that are synthesized in the plant and are responsible for the color of flower petals. The modification of pigment biosynthesis pathways using genetic engineering techniques can produce results that cannot be obtained by traditional breeding. This review presents the main advances in the application of genetic engineering techniques in floriculture using the example of flower color modification. There are several main areas of work with the genes of pigment biosynthesis. Among them, the strategy of suppressing gene expression is used most often. Expression of certain genes is suppressed to prevent pigment synthesis, or vice versa, to eliminate factors that hinder color development. The method of additional heterologous genes insertion to plants lacking them in the pathway of pigment biosynthesis is often used. Genomic editing, in particular by using the CRISPR/Cas system, is also used for color modification, but the application of this method to ornamental plants is a relatively recent innovation. Despite the rapid development of biotechnology, there are obstacles to the distribution of genetically modified plants on the world market. By addressing a number of problems, the production of transgenic ornamental plants may become economically more cost-effective and attractive than the development of new varieties exclusively through traditional breeding methods.

Quantification of petal patterns and colours of genus Sinningia (GESNERIACEAE)

Plants belonging to genus Sinningia are popular ornamental plants with diverse petal patterns and fascinating petal colours. The patterns and colours of petals are crucial horticultural properties that determine a plant's commercial value. Generally, the patterns and colours of petals are evaluated by experienced horticulturalists. However, manual evaluation can be subjective and the decision criterion could fluctuate due to fatigue. This work proposed a method to automatically quantify the petal patterns and colours by investigating 11 species of Sinningia . The images of the ventral petal of flower specimens were captured using flatbed scanners. The regions of interest (ROI) were defined and segmented from ventral images. Subsequently, a fully convolutional network was applied to the ROI for automatically segmenting variegated parts (i.e., spots, strips, and plaques) from the background. The patterns and colours of petals were then analysed. The proposed method can be used for the automation of petal pattern rating, which generally is performed using naked-eye observations. • A method was proposed to quantify petal colours and patterns. • A fully convolutional network (FCN) was trained to segment patterns of petals. • The patterns were categorised into 3 categories: spots, strips, and plaques. • The method is tested on the ventral petals of 10 species of genus Sinningia. • This approach is objective and precise compared with the method in DUS test.

Diversification of petal monoterpene profiles during floral development and senescence in wild roses: relationships among geraniol content, petal colour, and floral lifespan

Wild roses store and emit a large array of fragrant monoterpenes from their petals. Maximisation of fragrance coincides with floral maturation in many angiosperms, which enhances pollination efficiency, reduces floral predation, and improves plant fitness. We hypothesized that petal monoterpenes serve additional lifelong functions such as limiting metabolic damage from reactive oxygen species (ROS), and altering isoprenoid hormonal abundance to increase floral lifespan. Petal monoterpenes were quantified at three floral life-stages (unopened bud, open mature, and senescent) in 57 rose species and 16 subspecies originating from Asia, America, and Europe, and relationships among monoterpene richness, petal colour, ROS, hormones, and floral lifespan were analysed within a phylogenetic context. Three distinct types of petal monoterpene profiles, revealing significant developmental and functional differences, were identified: Type A, species where monoterpene abundance peaked in open mature flowers depleting thereafter; Type B, where monoterpenes peaked in senescing flowers increasing from bud stage, and a rare Type C (8 species) where monoterpenes depleted from bud stage to senescence. Cyclic monoterpenes peaked during early floral development, whereas acyclic monoterpenes (dominated by geraniol and its derivatives, often 100-fold more abundant than other monoterpenes) peaked during floral maturation in Type A and B roses. Early-diverging roses were geraniol-poor (often Type C) and white-petalled. Lifetime changes in hydrogen peroxide (H2O2) revealed a significant negative regression with the levels of petal geraniol at all floral life-stages. Geraniol-poor Type C roses also showed higher cytokinins (in buds) and abscisic acid (in mature petals), and significantly shorter floral lifespan compared with geraniol-rich Type A and B roses. We conclude that geraniol enrichment, intensification of petal colour, and lower potential for H2O2-related oxidative damage characterise and likely contribute to longer floral lifespan in monoterpene-rich wild roses.

Flower color mutation caused by spontaneous cell layer displacement in carnation (Dianthus caryophyllus)

A change of layer arrangement of shoot apical meristem (SAM) organized by three cell layers (L1, L2 and L3) is thought to be one of the provocations of bud sport, which often induces changes in phenotypic colors in periclinal chimeras. This paper describes a cell layer rearrangement which is the cause of spontaneous flower color mutation by using two carnation (Dianthus caryophyllus L.) cultivars that are presumably periclinal chimeras, ‘Feminine Minami’ (deep pink flower) and its recessive sport ‘Tommy Minami’ (pinkish red flower). The genotype of the acyl-glucose-dependent anthocyanin 5-glucosyltransferase (AA5GT) which is responsible for the color change of red to pink, in each cell layer was deduced by genomic analysis using tissues originated from specific cell layer and investigation of partial petal color mutations. In the results, the genotype of the L1 of ‘Feminine Minami’ was heterozygous for functional AA5GT and non-functional AA5GT carrying retrotransposon Ty1dic1 (AA5GT-Ty1dic1), and its inner cell layer hid red flower genotype, whereas AA5GT-Ty1dic1 of the L1 of ‘Tommy Minami’ became homogenic in absence of the insertion of a new Ty1dic1. Our outcomes concluded that the L1 of ‘Tommy Minami’ harboring the recessive AA5GT alleles are attributed to the inner cell layer of ‘Feminine Minami’ possessing red flower genotype.

NEW INSIGHTS INTO GRAUSA: REINSTATEMENT, DISTRIBUTION AND AFFINITIES OF LOASA ACAULIS (LOASACEAE, LOASOIDEAE)

Gracias al estudio de la descripcion original, tipos, material del herbario, trabajo de campo y evidencia molecular, Loasa acaulis es restablecida, lectotipificada y transferida a Grausa. Loasa acaulis fue sinonimizada anteriormente con Loasa lateritia. Ahora consideramos a ambas como dos especies diferentes para las cuales se proporcionan una clave y descripciones detalladas. Las diferencias florales clave incluyen el tamano, forma, angulo y color de los petalos, la forma de las escamas de nectar y la posicion de los filamentos dorsales en las escamas. No se localizaron especimenes que mostraran morfologias florales intermedias entre ambas especies. Los estudios de microscopio electronico de barrido de semillas maduras muestran una fuerte afinidad entre las dos especies, ya que carecen tanto de las paredes periclinales externas como de la fenestracion incompleta que se encuentra en otros miembros de Grausa. Ademas, el cono hilar caracteristico de otras especies de Grausa, esta menos desarrollado en L. acaulis y G. lateritia. Se secuenciaron cinco loci (cuatro plastidiales y uno nuclear) para analisis moleculares y se obtuvieron filogenias bajo enfoques de Maxima Verosimilitud y Bayesianos. Tanto las filogenias plastidiales como las nucleares ubicaron a Loasa acaulis como hermana de Grausa lateritia, ambas lejanamente emparentadas con Blumenbachia y Loasa. Finalmente, el estudio de especimenes de herbario revelo que Loasa acaulis no es endemica de Chile como se pensaba anteriormente, sino que tambien se encuentra en Argentina.

Segregation of Flower Color and Eyespot in Althea

Althea (Hibiscus syriacus) is an ornamental shrub prized for its winterhardiness, flower colors, and unique flower forms, including single-flowered and double-flowered types. Although floral traits are most important for breeders of althea, little is known about their segregation patterns. The objective of this study was to determine segregation patterns in flower color, including eyespot, among hybrid seedlings of elite taxa. Over 4 years, more than 3100 flowering seedlings were produced for observation of F1, F2, and backcross families. For each plant, data were collected including presence of eyespot and petal body color (CIEL*a*b*) using a colorimeter. Recessive testcrosses and χ2 analyses were performed among three taxa (‘Buddha Belly’, ‘Diana’, and White Chiffon®), and between this recessive group and a suite of colorful taxa. Self-pollination and intercrosses within homozygous dominant and homozygous recessive groups further confirmed their genotypes. Based on these results, we propose that eyespot is controlled by a single gene called spotless, named for the recessive allele that results in a complete elimination of color in flowers. Crosses that resulted in seedlings that all produced eyespots were observed to segregate for color in the petal body. Of these, one group produced white to blush pink petals, which was recessive to full color. Recessive testcrosses and χ2 analyses were performed among nine taxa exhibiting eyespots with white to blush petal bodies, and between taxa with full-color petal bodies. These testcrosses resulted in a putative homozygous dominant group composed mostly of blue and dark pink taxa, whereas the heterozygous group was composed mostly of pink taxa. Spotless taxa were also added to these two groups, suggesting an epistatic interaction with the spotless allele. Based on these results, we propose that petal body color is controlled by a single gene called geisha, named for the recessive allele that produces white to blush-pink petal bodies and dark red eyespot. This trait exhibits incomplete dominance and is under epistatic control by spotless. Geisha-type flowers lack pigment in the petal body, or exhibit a blush pink, likely produced by low levels of cyanidin, peonidin, and pelargonidin. The interaction and segregation of these two genes was confirmed in F1, F2, and backcross families from two crosses: Lil’ Kim™ × Blue Chiffon™ and Fiji™ × White Chiffon®. This study on segregation of flower color in H. syriacus contributes substantial and useful information on inheritance of color and will facilitate targeted breeding to improve this vibrant ornamental shrub.

Geographic Variation in Floral Color and Reflectance Correlates With Temperature and Colonization History.

Petal color variation within species is common and may be molded by abiotic or biotic selection pressures, or neutral population structure. For example, darker flowers may be favored in cooler environments because they absorb more solar radiation, elevating the temperature of reproductive structures. Additionally, flower color may evolve to attract the dominant or most efficient pollinator type in a given population. Here, we evaluate geographic variation in petal coloration across the range of Campanula americana in Eastern North America and test whether color covaries with abiotic factors, the pollination community, and genetic structure established through post-glacial expansion. Consistent with other studies, flowers from cooler, higher latitude populations were less reflective across the UV-NIR spectrum than those from warmer populations. Local temperature explained variation in petal reflectance better than the pollinator community or colonization history. Petal color perceived by trichromatic bee pollinators displayed a strong longitudinal pattern but was unassociated with climatic factors and the pollinator community. Instead, pollinator-perceived color was tightly correlated with the geographic distance from C. americana's glacial refugium. In total, abiotic conditions appear to shape large-scale geographic variation in the intensity of petal reflectance while genetic structure is the strongest driver of pollinator-perceived petal coloration. This study highlights the importance of abiotic factors and historical processes associated with range expansion as major evolutionary forces shaping diversity of flower coloration on large geographic scales.

Breeding of Standard Rose ‘Pinky Luna’ with a Fragrance, and Pastel Color of Pale Pink for Cut Flowers

A new Rosa cultivar ‘Pinky Luna’ was bred at Jeollabuk-do Agricultural Research and Extension Services. In the year 2011, between ‘Rose Yumi’ of white color as a mother plant and ‘Ocean Song’ of light purple color as a father plant were artificially crossed, and 87 seeds were obtained, subsequently 66 seedling individuals were raised during the course of the next year. The ‘R111953-25’ line, which possessed pristine characteristics such as a beautiful flower shape and petal color, a fragrance, and good productivity, was selected via the characteristic trial during the years 2012 - 2013, then finally named as ‘Pinky Luna’ after investigating the characteristics, the preference, and the productivity during the years 2014 - 2015. ‘Pinky Luna’ cultivar was a standard type for cut flower with the pastel color of pale pink (petal’s surface - pinkish white, WN155C (RHS color chart), petal’s back – very pale purple, RP69D) and a sweet fragrance within the flower. The new bred cultivar possessed several morphological characteristics including medium elliptical leaf shape, a deep green (N137A) leaf color, circular flower shape, and a broad oval petal type. The flower width and height of ‘Pinky Luna’ was larger than the control cultivar ‘Ocean Song’ at 10.5 cm, and 4.1 cm. respectively, and the number of its petals were far more than those at 67.0, and in addition, the diameter of petal was wider. The cut flower length and leaf growth of ‘Pinky Luna’ were smaller than the control cultivar, however, the annual amount of average cut flowers per square of the new cultivar were 157.6, which was 10.2 more stems than ‘Ocean Song’. Vase l ife lasted longer for a period of 11.7 days, and additionally the consumer preference of cut flowers was higher than that of ‘Ocean Song’.

Interspecific hybrid confirmation in wide crosses of cotton (Gossypium spp) through morphological, cytological and molecular analysis

Interspecific triploid hybrids were developed between tetraploid cultivated species Gossypium barbadense cv. Suvin and TCB 37 and diploid wild species Gossypium armourianum. The F1 hybridity was confirmed by morphological, cytological and molecular approaches. The ploidy level of interspecific F1 hybrid was triploid and completely male sterile. Female parents Suvin and TCB 37 had erect growth habit, yellow petals, palmate leaves, green stem, thick and prominent leaf veins, but Suvin had dense yellow anthers while TCB 37 was with dense creamy anthers whereas male parent has a spreading growth habit, yellow petals, cordate leaves, reddish green stem, medium dense yellow anthers, thin leaf veins, protruded stigma and glabrous plant body. The growth habit, petal colour, leaf shape and size of interspecific F1 hybrid were intermediate. Plant stem colour, hairyness, leaf pubescence and anther colour of Gossypium armourianum were observed to be dominant as hybrid fully resembled Gossypium armourianum for these characters. Petal spot was observed in both male and female parents but F1 hybrids had different intensity of petal spot in their petals. Variable expression of petal spot, anther colour and filament colour was observed in the F1 hybrids. Gossypium barbadense cv. Suvin and TCB 37 had 52 chromosomes, Gossypium armourianum had 26 chromosomes and the interspecific F1 was with 39 chromosomes. Significant differences were observed between pollen size, pollen fertility of parents and their hybrids. The F1 interspecific hybrid having more than 97% of sterile pollen grains. Out of 6 polymorphic SSR markers between parents, 3 markers unambiguously confirmed the hybrid status of interspecific hybrid. This hybrid may serve as useful genetic resource for the transfer of jassid resistance gene to Gossypium barbadense cultivars.

Floral pigment-scent associations in eight cut rose cultivars with various petal colors

The associations between petal pigments and scent compounds of Rosa hybrida were determined in eight commercial cultivars with different petal-colors, visually classified as four petal-color groups: ‘Beast’ and 'Marilyn Monroe' in white (W), ‘Good Time’ and ‘Penny Lane’ in yellow (Y), ‘Ahoi’ and ‘Pink Condor’ in pink (P), and ‘Pearl Red’ and ‘Vital’ in red (R). The petal pigments were distinct by visual petal color polymorphs. Yellow petals contained only carotenoids and unusually large amounts of β-carotenes as their primary pigments, whereas white ones included minimal levels of carotenoids. As expected, pink and red flowers had cyanin as the major anthocyanin. The amount of cyanin in petals was significantly higher in the red petals. Unlike petal pigments, the scents were abundant in white and yellow flowers, containing both phenolic and terpene compounds. The pink and red flowers, on the other hand, were scented with only phenolics as represented by 2-phenylethanol and 3,5-dimethoxytoluene, which existed in all eight cultivars. The amount of detected scent was up to six times higher in the white or yellow flowers than in the pink or red ones. A principal component analysis (PCA) with all detected pigments and scents resulted in classifying eight cultivars as three independent groups: (GI) ‘Beast’, (GII) ‘Good Time’, ‘Penny Lane’, and ‘Marilyn Monroe’, and (GIII) ‘Ahoi’, ‘Pink Condor’, ‘Pearl Red’, and ‘Vital’. ‘Marilyn Monroe’ (W) belonged to GII with yellow cultivars, not to GI with ‘Beast’ (W). The PCA coordinates of pink and red cultivars were so close that they belonged to the same group, GIII. With this information, we know that rose petal colors may differ in terms of their pigment and scent profiles.

Chromosome analysis of Hibiscus rosa-sinensis using CHIAS IV software

Hibiscus rosa-sinensis is a plant that has flowers with many variations in size, petal shape, petal colors, and chromosome number. Chromosome number and morphology information is the key to study its flower morphology and physiology. Chromosome Image Analyzing System IV (CHIAS IV) is software that used for mapping and identifying chromosome automatically. This report presents the results of chromosome analysis using CHIAS IV from four H. rosa-sinensis flower variations (single small red, single small pale red, single big red, and double red). Chromosome number and condensation patterns of individual chromosomes analysis suggested that these flower variations of H. rosa-sinensis exhibited polyploidy, with the smallest number found in single small pale red and the biggest number in single big red flower. CHIAS IV software is an effective tool to analyze chromosome image, especially to predict chromosome number, chromosome type, relative length, and condensation patterns of individual chromosome.

Flora of Usina São José, Igarassu, Pernambuco (Brazil): Lythraceae J. St.-Hil. and Onagraceae Juss

Lythraceae comprises about 30 genera and 600 species widely distributed in tropical and subtropical regions, while Onagraceae comprises 22 genera and about 660 species distributed in temperate and subtropical areas, predominantly in Americas. The aim of this work was to realize the taxonomic study of Lythraceae and Onagraceae species from Usina São José, Igarassu, Pernambuco (Brazil), an Atlantic forest remnant. Fieldworks and visits to herbaria HST, HUEFS, IPA, JPB, PEUFR and UFP were conducted between 2018 and 2019. Three Lythraceae species were recognized: Cuphea carthagenensis (Jacq.) J.F. Macbr., C. flava Spreg. and Rotala ramosior (L.) Koehne, and three Onagraceae species: Ludwigia erecta (L.) H. Hara, L. hyssopifolia (G. Don) Exell and L. octovalvis (Jacq.) P.H. Raven. Rotala ramosior is a new record for Brazil. Morphological descriptions, an identification key, phenological period, geographic distribution, taxonomic notes, photographs and illustrations of diagnostic characters are presented herein. The relevant morphological characters for species identification are phyllotaxy, indumentum of stems, shape and indumentum of leaves, petals color and apex, capsule shape, seeds arrangement and raphe size.

Morphological characterization of Damask rose (Rosa × damascena Herrm.) germplasm to select superior accessions

Damask rose (Rosa × damascena Herrm.) is the most important scented rose species, which some of its varieties are great importance for rose oil production. In the present survey, a total of 327 accessions of wild Damask rose from 21 geographically distinct regions of Iran were collected and transplanted in a protected conservation garden. After 2 years of plants establishment, phenotypic variation of accessions were evaluated using 36 morphological traits. High level of phenotypic variations was detected among the accessions especially desirable variations for commercial characters such as petal color, number of petals, and fragrance. The highest variability corresponded to petal number, leaf color, leaf stipule, and prickle shape. Flowering date varied from 08 May to 03 June. The petal number ranged between 17 and 159. The highest values for flower weight and petal weight were 5.67 and 4.61 g, respectively. Leaflet length ranged from 20.09 to 60.73 mm, while leaf width varied from 15.26 to 44.75 mm. Regression analysis indicated the importance of petal color, flowering date, and leaflet width/length in influencing floral scent. Dispersion diagram based on the first two principal components (40.86% of total variance) showed that the studied accessions were distributed into the plot. Some of the superior accessions were separated from others in scatter plot and characterized by the highest value of flower weight, petal weight, and petal number. In conclusion, the genotype collected will be useful for ex situ conservation and utilization in breeding programs of Damask rose.

Transcriptome analysis and metabolic profiling reveal the key role of carotenoids in the petal coloration of Liriodendron tulipifera

Liriodendron tulipifera, also known as tuliptree, is a popular ornamental horticultural plant with extraordinary tulip-shaped flowers characterized by an orange band near their base. The mechanisms underlying petal band-specific pigmentation during L. tulipifera flower development are unclear. Here, we combined nontargeted and targeted metabolomics and transcriptomics to identify a pathway cascade leading to carotenoid biosynthesis that is specifically activated in the petal band. The comparative analysis of carotenoid metabolites between L. tulipifera and Liriodendron hybrids indicates that γ-carotene, a rare carotene in plants, is the most likely orange pigment responsible for the coloration of the petal band. Phenotypic and transcriptomic analyses of developing petals reveal that the band area is first predefined by the loss of green color. Later, the band is maintained by locally activating and repressing carotenoid and chlorophyll biosynthesis genes, respectively. Two rate-limiting genes of carotene biosynthesis, carotenoid isomerase (CRTISO) and epsilon lycopene cyclase (ε-LCY), encode the core enzymes responsible for petal band-specific orange pigmentation in L. tulipifera. In particular, a putative additional ε-LCY copy specific to L. tulipifera may contribute to the distinct petal coloration pattern, compared with L. chinense. Taken together, our work provides a first glimpse of the metabolome and transcriptome dynamics in tuliptree flower coloration and provides a valuable resource for flower breeding or metabolic engineering as well as for understanding flower evolution in an early woody angiosperm.

Effects of Exogenous Abscisic Acid (ABA) on Carotenoids and Petal Color in Osmanthus fragrans 'Yanhonggui'.

Osmanthus fragrans is a well-known native plant in China, and carotenoids are the main group of pigments in the petals. Abscisic acid (ABA) is one of the products of the metabolic pathway of carotenoids. Application of ABA could affect pigmentation of flower petals by changing the carotenoid content. However, little is known about the effects of ABA treatment on carotenoid accumulation in O. fragrans. In this study, different concentrations of ABA (0, 150 and 200 mg/L) were spread on the petals of O. fragrans ‘Yanhonggui’. The petal color of ‘Yanhonggui’ receiving every ABA treatment was deeper than that of the control. The content of total carotenoids in the petals significantly increased with 200 mg/L ABA treatment. In the petals, α-carotene and β-carotene were the predominant carotenoids. The expression of several genes involved in the metabolism of carotenoids increased with 200 mg/L ABA treatment, including PSY1, PDS1, Z-ISO1, ZDS1, CRTISO, NCED3 and CCD4. However, the transcription levels of the latter two carotenoid degradation-related genes were much lower than of the five former carotenoid biosynthesis-related genes; the finding would explain the significant increase in total carotenoids in ‘Yanhonggui’ petals receiving the 200 mg/L ABA treatment.

CcMYB6-1 and CcbHLH1, two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis in cornflower

Anthocyanins in cornflower (Centaurea cyanus) is catalysed by a set of biosynthesis genes, however, the potential mechanism of transcriptional regulation remains unclear. In the present study, we traced the dynamic changes of petal colour development from white to violet and finally to blue on the same petal in cornflower. Pigment analysis showed that anthocyanin accumulation dramatically increased with petal colour development. Subsequently, nine libraries from above three colour regions were constructed for RNA-seq and 105,506 unigenes were obtained by de novo assembling. The differentially expressed genes among three colour regions were significantly enriched in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways, leading to the excavation and analysis of 46 biosynthesis genes involved in this process. Furthermore, four R2R3-CcMYBs clustered into subgroup 4 or subgroup 6 and one CcbHLH1 clustered into IIIf subgroup were screened out by phylogenetic analysis with Arabidopsis homologues. The promoters of flavanone 3-hydroxylase (CcF3H) and dihydroflavonol 4-reductase (CcDFR) were further isolated to investigate upstream regulation mechanism. CcMYB6-1 significantly upregulated the activity of above two promoters and stimulated anthocyanin accumulation by dual luciferase assay and transient expression in tobacco leaves, and its activity was obviously enhanced when co-infiltrated with CcbHLH1. Moreover, both yeast two-hybrid and bimolecular fluorescence complementation assays indicated the protein-protein interaction between these two activators. Based on these obtained results, it reveals that CcMYB6-1 and CcbHLH1 are two novel transcription factors synergistically involved in regulating anthocyanin biosynthesis. This study provides insights into the regulatory mechanism of anthocyanin accumulation in cornflower.

Analysis of flavonoids and anthocyanin biosynthesis-related genes expression reveals the mechanism of petal color fading of Malus hupehensis (Rosaceae)

Malus hupehensis (Pamp.) Rehder is an indigenous wild crabapple tree from China that has received increased attention, due to its significant ornamental and economical values. The flowers change from red to white during development, a phenomenon that is prevalent within Malus genus. However, there are no reports on the phytochemical aspects of M. hupehensis flower. Here, we investigated the mechanism of color fading through biochemical and molecular analysis of M. hupehensis petals during development. Sixteen flavonoids were detected, and cyanidin-3-galactoside was the major anthocyanin in petals. The levels of most compounds initially increased and then decreased during flower development. The changing profile of total anthocyanins was consistent with the changes in floral color. Analysis of anthocyanin biosynthesis-related genes showed that the expression levels of PAL, CHS, CHI, DFR, FLS, ANS, UFGT, MYB10 and MYB12 were significantly higher at early stages than at later stages, similarly to the change in cyanidin-3-galactoside contents. MYB10 and MYB12 significantly positively correlated with CHS, CHI and DFR. There was a particularly strong positive correlation between MYB10 and ANS, and MYB12 was strongly positively correlated with LAR. Thus, MYB10 appeared to regulate mainly anthocyanin biosynthesis, and MYB12 could influence the expression of multiple structural genes in both the anthocyanin and flavanol pathways. By contrast, MYB6, bHLH33 and WD40 were significantly and positively correlated with one another but negatively correlated with the levels of many compounds and most structural genes, indicating that these three transcription factors could regulate together anthocyanin biosynthesis. Our results revealed the information about the temporal and spatial regulation of anthocyanin biosynthesis in M. hupehensis and provide the diverse flower color intensities and patterning in Chinese crabapple.

Isolation of PlANS and PlDFR genes from herbaceous peony (Paeonia lactiflora Pall.) and its functional characterization in Arabidopsis and tobacco

Flower colour, which is largely determined by anthocyanin, is an important ornamental value of herbaceous peony (Paeonia lactiflora Pall.). Anthocyanin synthesis genes encode enzymes acting at different stages of flavonoid biosynthetic pathway. In this research, we cloned anthocyanin synthesis genes PlANS and PlDFR from red dominated herbaceous peony cultivar ‘Hongyanzhenghui’ and explore their functionality with respect to colour formation by genetic engineering techniques. The over-expression vectors pB1301-PlANS and pB1301-PlDFR were constructed and transformed as heterologous genes into Arabidopsis thaliana (A. thaliana) and tobacco (K326). The quantitative real-time PCR results showed the over expression of PlANS and PlDFR in transgenic plants from 10 to 1600 times. Phenotypic observation and HPLC detection at OD 525 nm also revealed the elevated anthocyanin content in the transgenic Arabidopsis. The a* and c* value of transgenic flower increased 3–4 times compared with control tobacco which are consistent with the observed colour change. Taken together, our findings showed that PlANS, PlDFR is participated in the anthocyanin biosynthesis pathway which could provide a theoretical basis for further research of flower colour in herbaceous peony. Our results provided an important research work of two P. lactiflora genes which are related to the red petal colour formation. It provides a theoretical basis for further identification of flower colour-related gene function study in P. lactiflora.