Accumulation Of Delphinidin Research Articles

Transcriptome analysis reveals biosynthesis and regulation of flavonoid in common bean seeds during grain filling

The Andean domesticated common beans (Phaseolus vulgaris) are significant sources of phenolic compounds associated with health benefits. However, the regulation of biosynthesis of these compounds during bean seed development remains unclear. To elucidate the gene expression patterns involved in the regulation of the flavonoid pathway, we conducted a transcriptome analysis of two contrasting Chilean varieties, Negro Argel (black bean) and Coscorron (white bean), at three developmental stages associated with seed color change, as well as different flavonoid compound accumulations. Our study reveals that phenolic compound synthesis initiates during seed filling, although it exhibits desynchronization between both varieties. We identified 10,153 Differentially Expressed Genes (DEGs) across all comparisons. The KEGG pathway ‘Flavonoid biosynthesis’ showed enrichment of induced DEGs in Negro Argel (PV172), consistent with the accumulation of delphinidin, petunidin, and malvidin hexosides in their seeds, while catechin glucoside, procyanidin and kaempferol derivatives were predominantly detected in Coscorrón (PV24). Furthermore, while the flavonoid pathway was active in both varieties, our results suggest that enzymes involved in the final steps, such as ANS and UGT, were crucial, inducing anthocyanin formation in Negro Argel. Additionally, during active anthocyanin biosynthesis, the accumulation of reserve proteins or those related to seed protection and germination was induced. These findings provide valuable insights and serve as a guide for plant breeding aimed at enhancing the health and nutritional properties of common beans.

Anthocyanin metabolic engineering of Euphorbia pulcherrima: advances and perspectives.

The range of floral colors is determined by the type of plant pigment accumulated by the plant. Anthocyanins are the most common flavonoid pigments in angiosperms; they provide a wide range of visible colors from red-magenta to blue-purple, products of cyanidin and delphinidin biosynthesis, respectively. For the floriculture industry, floral color is one of the most important ornamental characteristics for the development of new commercial varieties; however, most plant species are restricted to a certain color spectrum, limited by their own genetics. In fact, many ornamental crops lack bluish varieties due to the lack of activity of essential biosynthetic enzymes for the accumulation of delphinidin. An example is the poinsettia (Euphorbia pulcherrima Willd. ex Klotzsch), the ornamental plant symbol of Christmas and native to Mexico. Its popularity is the result of the variety of colors displayed by its bracts, a kind of modified leaves that accumulate reddish pigments based mainly on cyanidin and, to a lesser extent, on pelargonidin. The commercial success of this plant lies in the development of new varieties and, although consumers like the typical red color, they are also looking for poinsettias with new and innovative colors. Previous research has demonstrated the possibility of manipulating flower color through metabolic engineering of the anthocyanin biosynthesis pathway and plant tissue culture in different ornamental plant species. For example, transgenic cultivars of flowers such as roses, carnations or chrysanthemums owe their attractive bluish colors to a high and exclusive accumulation of delphinidin. Here, we discuss the possibilities of genetic engineering of the anthocyanin biosynthetic pathway in E. pulcherrima through the introduction of one or more foreign delphinidin biosynthetic genes under the transcriptional control of a pathway-specific promoter, and the genome editing possibilities as an alternative tool to modify the color of the bracts. In addition, some other approaches such as the appropriate selection of the cultivars that presented the most suitable intracellular conditions to accumulate delphinidin, as well as the incorporation of genes encoding anthocyanin-modifying enzymes or transcription factors to favor the bluish pigmentation of the flowers are also revised.

Flower color modification through co-overexpression of the VtF3′5′H and RhNHX genes in Rosa hybrida

Roses (Rosa hybrida) are a highly merchandised flower but lack blue varieties. Overexpression of the flavonoid 3′,5′-hydroxylase (F3′5′H) gene can increase the accumulation of blue pigment (delphinidin anthocyanin). However, sometimes the effect of F3′5′H gene alone is inadequate for producing blue flowers. Furthermore, the internal environment of the cell, such as an increase in pH, can also help the conversion of anthocyanins to blue pigments. Nonetheless, genetic engineering methods can simultaneously introduce multiple genes at the same time to regulate the development of blue pigments to achieve the ultimate breeding goal of producing blue color in roses. In the present study, to simultaneously adjust the accumulation of delphinidin and vacuolar pH, we introduced the Viola tricolor flavonoid 3′,5′-hydroxylase (VtF3′5′H) and Rosa hybrida Na+/H+ exchanger (RhNHX) genes into the white rose line “KR056002” using Agrobacterium-mediated transformation. The quantitative real time polymerase chain reaction (qRT-PCR) results showed that the heterologous genes in the transgenic lines were highly expressed in petals and leaves, and simultaneously promoted the expression of related anthocyanin synthesis structural genes. Obvious color changes were observed in both petals and young leaves, especially when petals changed from white to red-purple. The formation of delphinidin was not detected in the petals of control plants, whereas the petals of transgenic lines had higher delphinidin content (135–214 μg/l) and increased pH value (0.45–0.53) compared with those of control plants.

Integrated transcriptome and metabolome analysis reveals the anthocyanin biosynthesis mechanisms in blueberry (Vaccinium corymbosum L.) leaves under different light qualities.

Blueberry (Vaccinium corymbosum L.) is a popular fruit with an abundance of anthocyanins in its leaves and fruits. Light is one of the pivotal environmental elements that affects plant growth and development, but the regulatory mechanism between light quality and anthocyanin formation is poorly understood. An integrated transcriptome and metabolome analysis was performed to investigate the effects of white (control), blue (B), red (R), and red/blue (60R/40B) light on blueberry growth and reveal the potential pathway controlling anthocyanin biosynthesis in blueberry leaves. The anthocyanin content was significantly improved by the blue and red/blue light when compared with white light, whereas there was a significant reduction in the photosynthesis under the blue light, showing an inverse trend to that of anthocyanin accumulation. Transcriptomic analysis resulted in the assembly of 134,709 unigenes. Of these, 22 were differentially expressed genes (DEGs) that participate in the anthocyanin biosynthesis pathway, with the majority being significantly up-regulated under the blue light. Most of the photosynthesis-related genes that were down-regulated were expressed during anthocyanin accumulation. Targeted metabolome profiling identified 44 metabolites associated with anthocyanin biosynthesis. The contents of most of these metabolites were higher under blue light than the other light conditions, which was consistent with the transcriptome results. The integrated transcriptome and metabolome analysis suggested that, under blue light, leucoanthocyanidin dioxygenase (LDOX), O-methyltransferase (OMT), and UDP-glucose flavonoid glucosyltransferase (UFGT) were the most significantly expressed, and they promoted the synthesis of cyanidin (Cy), malvidin (Mv), and pelargonidin (Pg) anthocyanidins, respectively. The expression levels of dihydroflavonol 4-reductase (DFR) and OMT, as well as the accumulation of delphinidin (Dp), peonidin (Pn), and petunidin (Pt), were significantly increased by the red/blue light. The blue and red/blue lights promoted anthocyanin biosynthesis via inducing the expression of key structural genes and accumulation of metabolites involved in anthocyanin synthesis pathway. Moreover, there was a possible feedback regulating correlation between anthocyanin biosynthesis and photosynthesis under different light qualities in blueberry leaves. This study would provide a theoretical basis for elucidating the underlying regulatory mechanism of anthocyanin biosynthesis of V. corymbosum.

Dissecting the impact of environment, season and genotype on blackcurrant fruit quality traits

This work aims to determine the effect of genotype x environment (GxE) interaction that influence blackcurrant (Ribes nigrum) fruit quality. We applied metabolomics-driven analysis on fruits from four cultivars grown in contrasting European-locations over two seasons. By integrating metabolomics and sensory analysis, we also defined specific metabolic signatures associated with consumer acceptance. Our results showed that rainfall is a crucial factor associated with accumulation of delphinidin- and cyanidin-3-O-glucoside, the two mayor blackcurrant pigments meanwhile temperature affects the main organic acid levels which can be decisive for fruit taste. Sensorial analysis showed that increases in terpenoid and acetate ester volatiles were strongly associated with higher appreciation score, while proacacipetalin, a cyanogenic-glycoside, was positively associated to bitter taste. Our results pave the way for the selection of high-quality cultivars and suitable production sites for blackcurrant cultivation.

Selection of suitable lily cultivars by using needle agroinfiltration for blue flower production

ABSTRACT Lily is one of the most important flower crops that it lacks blue-purple flowers due to the absence of the flavonoid 3’,5’-hydroxylase gene and deficiency of delphinidin accumulation. In this study, the possibility of transient expression of F3ʹ5’H gene and delphinidin accumulation were examined by agroinfiltration method in petals of six commercial lily cultivars. Three sizes of the flower buds and new blossomed flowers were harvested and measured for anthocyanidin content by the high-performance liquid chromatography to determine the appropriate stage for agroinfiltration. Then, the petals were infiltrated at the best stage with two vectors namely pBI121-35S carrying only F3ʹ5’H and pBIH-35S-Del2 harbouring F3ʹ5’H, DFR and ANS genes. The results indicated that the largest buds and new blossomed flowers showed the highest contents of anthocyanins in all subjected cultivars. Therefore, new blossomed flowers were injected with two different expression vectors. The HPLC analyses showed the highest delphinidin contents were detected in the two cultivars ‘Brindisi’ and ‘Gaucho’ by 77.63 and 43.60 μg g−1 fresh weights, respectively, when they were infiltrated with the pBIH-35S-Del2 construct. Real-Time PCR confirmed the results of anthocyanin measurements properly. Consequently, these two cultivars were selected as the appropriate hosts for lily stable transformation programmes. Abbreviations: ANS: Anthocyanidin synthase; CaMV: Cauliflower mosaic virus; CHS: Chalcone synthase; CTAB: Cetyl trimethyl ammonium bromide; DFR: Dihydroflavonol 4-reductase; DHK: Dihydrokaempferol; DHM: Dihydromyricetin; F3ʹH: Flavonoid 3’-hydroxylase; F3ʹ5’H: Flavonoid 3’, 5’-hydroxylase; FW: Fresh weight; GUS: β-glucuronidase; HPLC: High performance liquid chromatography; OD: Optical density; T-DNA: Transfer deoxyribonucleic acid.

MYB transcription factors GmMYBA2 and GmMYBR function in a feedback loop to control pigmentation of seed coat in soybean.

Soybean has undergone extensive selection pressures for seed nutrient composition and seed color during domestication, but the major genetic loci controlling seed coat color have not been completely understood, and the transcriptional regulation relationship among the loci remains elusive. Here, two major regulators, GmMYBA2 and GmMYBR, were functionally characterized as an anthocyanin activator and repressor, respectively. Ectopic expression of GmMYBA2 in soybean hairy roots conferred the enhanced accumulation of delphinidin and cyanidin types of anthocyanins in W1t and w1T backgrounds, respectively, through activating anthocyanin biosynthetic genes in the reported loci. The seed coat pigmentation of GmMYBA2-overexpressing transgenic plants in the W1 background mimicked the imperfect black phenotype (W1/w1, i, R, t), suggesting that GmMYBA2 was responsible for the R locus. Molecular and biochemical analysis showed that GmMYBA2 interacted with GmTT8a to directly activate anthocyanin biosynthetic genes. GmMYBA2 and GmMYBR might form a feedback loop to fine-tune seed coat coloration, which was confirmed in transgenic soybeans. Both GmTT8a and GmMYBR that were activated by GmMYBA2 in turn enhanced and obstructed the formation of the GmMYBA2-GmTT8a module, respectively. The results revealed the sophisticated regulatory network underlying the soybean seed coat pigmentation loci and shed light on the understanding of the seed coat coloration and other seed inclusions.

Assessment of violet-blue color formation in Phalaenopsis orchids

BackgroundPhalaenopsis represents an important cash crop worldwide. Abundant flower colors observed in Phalaenopsis orchids range from red-purple, purple, purple-violet, violet, and violet-blue. However, violet-blue orchids are less bred than are those of other colors. Anthocyanin, vacuolar pH and metal ions are three major factors influencing flower color. This study aimed to identify the factors causing the violet-blue color in Phalaenopsis flowers and to analyze whether delphinidin accumulation and blue pigmentation formation can be achieved by transient overexpression of heterologous F3’5’H in Phalaenopsis.ResultsCyanidin-based anthocyanin was highly accumulated in Phalaenopsis flowers with red-purple, purple, purple-violet, and violet to violet-blue color, but no true-blue color and no delphinidin was detected. Concomitantly, the expression of PeF3’H (Phalaenopsis equestrsis) was high, but that of PhF3’5’H (Phalaenopsis hybrid) was low or absent in various-colored Phalaenopsis flowers. Transient overexpression of DgF3’5’H (Delphinium grandiflorum) and PeMYB2 in a white Phalaenopsis cultivar resulted a 53.6% delphinidin accumulation and a novel blue color formation. In contrast, transient overexpression of both PhF3’5’H and PeMYB2 did not lead to delphinidin accumulation. Sequence analysis showed that the substrate recognition site 6 (SRS6) of PhF3’5’H was consistently different from DgF3’5’Hs at positions 5, 8 and 10. Prediction of molecular docking of the substrates showed a contrary binding direction of aromatic rings (B-ring) with the SRS6 domain of DgF3’5’H and PhF3’5’H. In addition, the pH values of violet-blue and purple Phalaenopsis flowers ranged from 5.33 to 5.54 and 4.77 to 5.04, respectively. Furthermore, the molar ratio of metal ions (including Al3+, Ca2+ and Fe3+) to anthocyanin in violet-blue color Phalaenopsis was 190-, 49-, and 51-fold higher, respectively, than those in purple-color Phalaenopsis.ConclusionCyanidin-based anthocyanin was detected in violet-blue color Phalaenopsis and was concomitant with a high pH value and high molar ratio of Al3+, Ca2+ and Fe3+ to anthocyanin content. Enhanced expression of delphinidin is needed to produce true-blue Phalaenopsis.

Effects of knocking out three anthocyanin modification genes on the blue pigmentation of gentian flowers

Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3′-O-glycosyltransferase (Gt3′GT), and anthocyanin 5/3′-aromatic acyltransferase (Gt5/3′AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3′GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3′AT resulted in the accumulation of delphinidin 3G-5G-3′G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3′GT and the other involving an acylation by 5/3′AT. The Gt5GT, Gt3′GT, and Gt5/3′AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3′-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanidin Biosynthesis of Grape Hyacinth.

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Comparative analysis of the quality and health-promoting compounds of two-shaped fruits of wild Lycium ruthenicum Murr. from the Qinghai–Tibet Plateau

Lycium ruthenicum Murr. is an econo-ecological tree species that abounds in the Qinghai–Tibet Plateau, which is located in the Chaka Salt Lake region of Qinghai Province, China. This study compared the phenotypes and main inclusions in two-shaped berries (flat peach and sphere types) at different developmental stages of L. ruthenicum Murr. to provide first-hand information for targeted breeding toward higher quality and stronger stress resistance of the fruit. Results showed the distinct advantage of the quality characters of flat peach-type fruits over the sphere-type with regard to the length of bearing shoot, internode, peduncle, single fruit weight, and 100-pod weight. In terms of inclusions, the anthocyanin contents in two-shaped fruits showed no significant difference at S5. Anthocyanin accumulated in the developing fruits, and the amount peaked at S4 and then gradually decreased. The expression profiles of the key genes in anthocyanin biosynthesis were correlated with anthocyanin accumulation during fruit development, and the high expressions of flavonoid 3′,5′-hydroxylase gene and dihydroflavonol 4-reductase gene were found to be the critical factors of specific delphinidin accumulation. Sphere-type mature fruits had considerable higher total polyphenol level than flat peach fruits, but the opposite result was observed for polysaccharides. The contents of Ca, Co, Cr, Cu, K, Li, Mn, P, Ti, V, and Al presented no considerable differences, whereas B, Mg, Na, Si, and Zn exhibited evident advantages in sphere-type fruits. α-Toc was the most abundant component in both fruits and no evident difference in its content was observed. The data on nutritional contents provide a theoretical basis for different breeding goals of L. ruthenicum and its further utilization as health food.

The ROSEA1 and DELILA transcription factors control anthocyanin biosynthesis in Nicotiana benthamiana and Lilium flowers

The activity of anthocyanin biosynthesis genes is regulated at the transcriptional level, thus manipulation of transcription factors (TFs) is an ideal strategy to alter the expression of multiple target genes. In this study, we investigated the effect of introducing ROSEA1 (ROS1, a MYB-type) and DELILA (DEL, a bHLH-type) TFs from snapdragon under control of a flower specific promoter, Floral Binding Protein 1 (FBP1) from petunia into Nicotiana benthamiana flowers and Lilium tepals. The usefulness of the FBP1 promoter was demonstrated by the generation of purplish flowers in otherwise normal-growing plants of N. benthamiana, while the expression by the 35S promoter led to the development of stunted plants with anthocyanins in all parts. N. benthamiana was successfully transformed by ROS1 alone and by a combination of ROS1 + DEL. The observed accumulation of delphinidin corresponded to the expression of NbCHS, NbF3H, NbDFR and NbANS. The effect of ROS1 + DEL on Lilium flower colour was investigated using agroinfiltration. A higher cyanidin accumulation was observed in tepals of the Oriental hybrid lily cv. ‘Perth’, resulting in deeper pink colouration at the infiltrated area. Nevertheless, the introduction of ROS1 + DEL did not produce any phenotypic changes to the white-flowered L. longiflorum cv. ‘Lincoln’ and the white Oriental hybrid lily cv. ‘Rialto’ due to other deficiencies in their anthocyanin biosynthetic pathway. Co-expression of ROS1 + DEL under control of the FBP1 promoter together with active structural anthocyanin biosynthetic genes can result in modification of Lilium flower colour.

Agroinfiltration: a rapid and reliable method to select suitable rose cultivars for blue flower production.

Rose cultivars with blue flower color are among the most attractive breeding targets in floriculture. However, they are difficult to produce due to the low efficiency of transformation systems, interactive effects of hosts and vectors, and lengthy processes. In this study, agroinfiltration-mediated transient expression was investigated as a tool to assess the function of flower color genes and to determine appropriate host cultivars for stable transformation in Rosa hybrida. To induce delphinidin accumulation and consequently to produce blue hue, the petals of 30 rose cultivars were infiltrated with three different expression vectors namely pBIH-35S-CcF3'5'H, pBIH-35S-Del2 and pBIH-35S-Del8, harbouring different sets of flower color genes. The results obtained showed that the ectopic expression of the genes was only detected in three cultivars with dark pink petals (i.e. 'Purple power', 'High & Mora' and 'Marina') after 6-8days. The high performance liquid chromatography analyses confirmed delphinidin accumulation in the infiltrated petals caused by transient expression of CcF3'5'H gene. Moreover, there were significant differences in the amounts of delphinidin among the three cultivars infiltrated with the three different expression vectors. More specifically, the highest delphinidin content was detected in the cultivar 'Purple power' (4.67µgg-1 FW), infiltrated with the pBIH-35S-Del2 vector. The expression of CcF3'5'H gene in the infiltrated petals was also confirmed by real time PCR. In conclusion and based on the findings of the present study, the agroinfiltration could be regarded as a reliable method to identify suitable rose cultivars in blue rose flower production programs.

Effect of bearing position on phenolics profiles in the skins of four cultivars of grapevine (Vitis vinifera L.)

SummaryThe fan-shaped trellis training system, resulting in grapevines (Vitis vinifera L.) with more than two trunks, has been widely used in northern China. The fruiting zone of grapevines trained to the fan-shaped trellis system is distributed from the top to the bottom of the canopy. The phenolics profiles of the skins of ‘Cabernet Sauvignon’, ‘Merlot’, ‘Chardonnay’, and ‘Italian Riesling’ grape berries were analysed to measure the effect of differences in bearing position on the phenolic compound and anthocyanin compositions of grape berries.The results showed that the upper bearing position significantly increased the concentrations of most phenolic compounds in grape berry skins. Among the non-anthocyanin and anthocyanin phenolic compounds identified in this study, the upper bearing position promoted the accumulation of delphinidin-, cyanidin-, and petunidin-glucosides in the skin of the two red grape cultivars. However, lowering the bearing position was associated with greater methylation of anthocyanins, and the flow of photosynthate towards the biosynthesis of kaempferol. These results were correlated with micrometeorological alterations in the canopy at the different bearing positions. The findings of this study suggest that higher bearing positions can increase the accumulation of phenolic compounds in grape berry skins in four different grapevine cultivars.

Achievements and perspectives in biochemistry concerning anthocyanin modification for blue flower coloration.

Genetic engineering of roses and other plants of floricultural importance to give them a truly blue petal color is arguably one of the holy grails of plant biotechnology. Toward this goal, bluish carnations and roses were previously engineered by establishing an exclusive accumulation of delphinidin (Dp)-type anthocyanins in their petals via the heterologous expression of a flavonoid 3',5'-hydroxylase gene. Very recently, purple-blue varieties of chrysanthemums were also genetically engineered via a similar biochemical strategy. Although the floral colors of these transgenic plants still lack a true blue color, the basis for the future molecular breeding of truly blue flowers is via the engineering of anthocyanin pathways. Anthocyanins with multiple aromatic acyl groups (often referred to as polyacylated anthocyanins) in the 3'- or 7-position tend to display a more stable blue color than non-acylated anthocyanins. The 7-polyacylation process during the biosynthesis of purple-blue anthocyanins in delphinium (Delphinium grandiflorum) was found to occur in vacuoles using acyl-glucose as both the glucosyl and acyl donor. Glucosyltransferases and acyltransferases involved in anthocyanin 7-polyacylation in delphinium are vacuolar acyl-glucose-dependent enzymes belonging to the glycoside hydrolase family 1 and serine carboxypeptidae-like protein family, respectively. The 7-polyacylation proceeds through the alternate glucosylation and p-hydroxybenzoylation catalyzed by these enzymes. p-Hydroxybenzoyl-glucose serves as the p-hydroxybenzoyl and glucosyl donor to produce anthocyanins modified with a p-hydroxybenzoyl-glucose concatemer at the 7-position. This novel finding has provided a potential breakthrough for the genetic engineering of truly blue flowers, where polyacylated Dp-type anthocyanins are accumulated exclusively in the petals.

Violet/Blue Chrysanthemums—Metabolic Engineering of the Anthocyanin Biosynthetic Pathway Results in Novel Petal Colors

Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color.

Genetic Engineering of Novel Bluer-Colored Chrysanthemums Produced by Accumulation of Delphinidin-Based Anthocyanins

Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red-purple to a purple-violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums.

Flower-specific expression of the Phalaenopsis flavonoid 3′, 5′-hydoxylase modifies flower color pigmentation in Petunia and Lilium

Flavonoid 3′, 5′-hydoxylase (F3′5′H) is a key enzyme for biosynthesis of the blue anthocyanin pigment delphinidin. A number of F3′5′H genes from dicots have been tested for their effects on flower pigmentation; here F3′5′H from a monocot was tested for its effect on delphinidin accumulation in petals. To this end, F3′5′H (PhF3′5′H) from the orchid Phalaenopsis was expressed under the control of the chalcone synthase promoter in petunia flowers. Quantitative RT-PCR showed that PhF3′5′H was expressed mainly in the petal limb; this expression produced an increase in dihydromyricetin and delphinidin and a change in petal color from pink to deeper pink. To increase the accumulation of delphinidin, Hyacinth HyDFR, which encodes dihydroflavonol 4-reductase, and petunia DifF, which encodes a cytochrome b 5 that is required for full activity of F3′5′H were overexpressed. The HyDFR petunia transformants had a deeper color petal limb, increased dihydromyricetin and delphinidin contents and adaxial petals with a number of blue cells. The flowers of the DifF petunia transformants also showed a slight color change. We also tested PhF3′5′H in Lilium oriental ‘Sorbonne’, where transient PhF3′5′H expression by particle bombardment resulted in purple cells in the petals. Production of blue flowers by Phalaenopsis F3′5′H and hyacinth DFR potentially enables manipulation of flower color in ornamental plants, including production of blue flowers.

Climate Effects on Anthocyanin Accumulation and Composition in the Pomegranate (Punica granatumL.) Fruit Arils

Worldwide pomegranate (Punica granatum L.) production has expanded greatly due to recent evidence on the fruit health attributes. The fruit's unique red color, conferred by anthocyanins, is an imperative sensory quality. Climate effects on the fruit's internal color were reported earlier. The present study investigated the influence of a wide range of temperature regimes (∼7-40 °C) on pomegranates' aril anthocyanins. The study included two deciduous and two evergreen accessions as well as desert and Mediterranean orchards. RP-HPLC analysis of the arils' anthocyanins revealed mono- and diglucosylated delphinidins and cyanidins as the major anthocyanins and pelargonidins as minor components. Anthocyanin accumulation changed inversely to the season's temperatures. Cyanidins were generally more abundant but delphinidin accumulation was enhanced in cooler season. Monoglucosylated anthocyanins prevailed at cooler temperatures and subsided during seasonal warming with a concomitant increase in diglucoside proportion. The findings can benefit breeding and agricultural efforts to enhance pomegranate quality, especially in the face of "global warming".

Recent Progress of Flower Colour Modification by Biotechnology

Genetically-modified, colour-altered varieties of the important cut-flower crop carnation have now been commercially available for nearly ten years. In this review we describe the manipulation of the anthocyanin biosynthesis pathway that has lead to the development of these varieties and how similar manipulations have been successfully applied to both pot plants and another cut-flower species, the rose. From this experience it is clear that down- and up-regulation of the flavonoid and anthocyanin pathway is both possible and predictable. The major commercial benefit of the application of this technology has so far been the development of novel flower colours through the development of transgenic varieties that produce, uniquely for the target species, anthocyanins derived from delphinidin. These anthocyanins are ubiquitous in nature, and occur in both ornamental plants and common food plants. Through the extensive regulatory approval processes that must occur for the commercialization of genetically modified organisms, we have accumulated considerable experimental and trial data to show the accumulation of delphinidin based anthocyanins in the transgenic plants poses no environmental or health risk.