Flower Pigmentation Research Articles

Total synthesis of apigenin 7,4′-di- O-β-glucopyranoside, a component of blue flower pigment of Salvia patens, and seven chiral analogues

We have succeeded in the first total synthesis of apigenin 7,4′-di- O-β- d-glucopyranoside ( 1a ), a component of blue pigment, protodelphin, from naringenin ( 2 ). Glycosylation of 2 according to Koenigs–Knorr reaction provided a monoglucoside 4a in 80% yield, and this was followed by DDQ oxidation to give apigenin 7- O-glucoside ( 12a ). Further glycosylation of 4′-OH of 12a with 2,3,4,6-tetra- O-acetyl-α- d-glucopyranosyl fluoride ( 5a ) was achieved using a Lewis acid-and-base promotion system (BF 3·Et 2O, 2,6-di- tert-butyl-4-methylpyridine, and 1,1,3,3-tetramethylguanidine) in 70% yield, and subsequent deprotection produced 1a . Synthesis of three other chiral isomers of 1a , with replacement of d-glucose at 7 and/or 4′-OH by l-glucose ( 1b– d ), and four chiral isomers of apigenin 7- O-β-glucosides ( 6a, b ) and 4′- O-β-glucosides ( 7a, b ) also proved possible.

Genetics and epigenetics in flower pigmentation associated with transposable elements in morning glories

Among the genus Ipomoea, three morning glories, I. nil (the Japanese morning glory), I. purpurea (the common morning glory), and I. tricolor, were domesticated well for floricultural plants, and many spontaneous mutants displaying various flower pigmentation patterns were isolated. Most of these spontaneous mutations were found to be caused by the insertion of DNA transposable elements in the genes for the anthocyanin pigmentation in flowers, and many of them exhibited variegated flowers, such as white flowers with pigmented spots and sectors. Here, we describe the historical background of the mutants displaying variegated flowers and review the genetic and epigenetic regulation in flower pigmentation associated with transposable elements of these morning glories. The flecked, speckled, r-1, and purple mutations in I. nil were caused by insertions of Tpn1 and its relatives in the En/Spm superfamily, Tpn2, Tpn3, and Tpn4, into the genes for anthocyanin coloration in flowers, i.e., DFR-B, CHI, CHS-D, and InNHX1, respectively. Similarly, the flaked and pink mutants of I. purpurea have distantly related elements, Tip100 and Tip201, in the Ac/Ds superfamily inserted into the CHS-D and F3'H genes, respectively. The flower variegation patterns can be determined by the frequency and timing of the excision of these transposons, and their stable insertions produce plain color flowers without generating pigmented spots or sectors; furthermore, both genetic and epigenetic regulation appeared to play important roles in determining the frequency and timing of the excision of the transposons. However, flower variegation is not always associated with the excision of an integrated DNA transposon from one of the genes for anthocyanin pigmentation. The mutant Flying Saucers of I. tricolor displaying variegated flowers was found to have the transposon ItMULE1 inserted into the DFR-B promoter region, but no excision of ItMULE1 from the DFR-B could be detected in the variegated flower lines. The instable pearly-vrg allele in cv. Flying Saucers is likely to be an epiallele because the DNA methylation in the DFR-B promoter appeared to be associated with flower pigmentation.

Optimizing Marigold (Tagetes erecta L.) Petal and Pigment Yield

African marigold (Tagetes erecta L.) flower pigments can be extracted and used as a natural food additive to color egg yolks orange and poultry skin yellow. Five cultivars were examined for their ability to be grown commercially and mechanically harvested. ‘E‐1236’ was consistently a top producer for three seasons in terms of flower number, flower diameter, plant and flower canopy height, plant stand, and fresh flower, dried flower, and dried petal yield. E‐1236 produced the greatest quantity of lutein, a carotenoid pigment, in 1998 (22.0 kg ha−1), and E‐1236 and ‘Orange Lady’ both produced the greatest quantities in 1999 (20.7 and 21.3 kg ha−1, respectively). Transplanted rather than direct‐seeded plants produced two more harvests in a single season resulting in greater amounts of lutein production by transplants. In 1998, one mid‐season ammonium nitrate application (28 kg ha−1) resulted in larger flower diameters with direct‐seeded plants but did not affect dried petal yield. Plants were hedge trimmed in 1999 to mimic mechanical harvesting; this resulted in a 45 to 55% reduction in flower harvest data compared with hand‐harvested flowers. Of the cultivars tested, Orange Lady produced the greatest quantity of lutein (10.6 kg ha−1) when hedge trimmed.

Seasonal variation in pigmentation and anthocyanidin phenetics in commercial Eustoma flowers

The seasonal change in petal color and pigmentation of 29 commercial Eustoma cultivars was studied. The flowers are basically divided into four groups according to the major anthocyanidin phenotype in association with petal coloration, i.e., delphinidin (Dp)-based (purple flower), cyanidin (Cy)-based (reddish purple flower), pelargonidin (Pg)-based (pink flower), and none (white flower) groups. The constitution of petal anthocyanidins was not changed by forcing treatment in most of the flowers. Lightness ( L ∗ ) and chroma ( C ∗ , color saturation) showed a change along with the increase/decrease of hue angle difference (Δ H ∗ ), thus simultaneously the chromatic tonalities tended to move to redder and bluer, respectively. Floral pigment clustering described two flower groups in a dendrogram, based on anthocyanidin constitutions as phenetic markers, which are apparently the Dp- and Pg-based phenotypes of anthocyanidin syntheses. The Cy-based flowers made a subcluster with the Pg-based flowers, indicating a close relationship in the biosynthesis of the two anthocyanidins, and suggesting the Dp- and Pg-syntheses complement one another.

Seasonal variation in pigmentation and anthocyanidin phenetics in commercial Eustoma flowers

The seasonal change in petal color and pigmentation of 29 commercial Eustoma cultivars was studied. The flowers are basically divided into four groups according to the major anthocyanidin phenotype in association with petal coloration, i.e., delphinidin (Dp)-based (purple flower), cyanidin (Cy)-based (reddish purple flower), pelargonidin (Pg)-based (pink flower), and none (white flower) groups. The constitution of petal anthocyanidins was not changed by forcing treatment in most of the flowers. Lightness ( L ∗) and chroma ( C ∗, color saturation) showed a change along with the increase/decrease of hue angle difference (Δ H ∗), thus simultaneously the chromatic tonalities tended to move to redder and bluer, respectively. Floral pigment clustering described two flower groups in a dendrogram, based on anthocyanidin constitutions as phenetic markers, which are apparently the Dp- and Pg-based phenotypes of anthocyanidin syntheses. The Cy-based flowers made a subcluster with the Pg-based flowers, indicating a close relationship in the biosynthesis of the two anthocyanidins, and suggesting the Dp- and Pg-syntheses complement one another.

Daphnetin Methylation by a Novel O-Methyltransferase Is Associated with Cold Acclimation and Photosystem II Excitation Pressure in Rye

In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, (1)H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure.

Influence of glyphosate on flower morphogenesis and pigmentation in Petunia hybrida

Influence of glyphosate on flower morphogenesis and pigmentation in Petunia hybrida

SVISS – a novel transient gene silencing system for gene function discovery and validation in tobacco plants

We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family.

Inheritance of the An2 Gene and Epistatic Interactions in Petunia exserta × P. axillaris Hybrids

A regulatory gene, An2, controls structural genes within the flavonoid biosynthetic pathway. The inheritance of An2 expression in crosses between P. axillaris (an2) and P. exserta (An2+) was studied. Floral pigmentation was quantitatively inherited and involved the expression of a single regulatory gene (An2) and three structural genes (Hf1, An6 and Fl). White flowers were produced in an2- genotypes; while pigmented flowers were produced in An2+ genotypes. The intensity of pigmentation was determined by the interaction of An2 with An6, Hf1 and Fl, as well as substrate competition between the An6 and Fl encoded enzymes.

Duplication and adaptive evolution of the chalcone synthase genes of Dendranthema (Asteraceae).

Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids, which are important for the pigmentation of flowers and act as attractants to the pollinators. Genes encoding CHS constitute a multigene family in which the copy number varies among plant species and functional divergence appears to have occurred repeatedly. Plants of the Dendranthema genus have white, yellow, and pink flowers, exhibiting considerable variation in flower color. In this article, 18 CHS genes from six Dendranthema species were sequenced. Two of them were found to be pseudogenes. The functional Dendranthema CHS genes formed three well-supported subfamilies: SF1, SF2, and SF3. The inferred phylogeny of the CHS genes of Dendranthema and Gerbera suggests that those genes originated as a result of duplications before divergence of these two genera, and the function of Dendranthema CHS genes have diverged in a similar fashion to the Gerbera CHS genes; i.e., the genes of SF1 and SF3 code for typical CHS enzymes expressed during different stages of development, whereas the genes of SF2 code for another enzyme that is different from CHS in substrate specificity and reaction. Relative rate tests revealed that the Dendranthema CHS genes significantly deviated from clocklike evolution at nonsynonymous sites. Maximum likelihood analysis showed that the nonsynonymous-synonymous (omega = d(N)/d(S)) rate ratio for the lineage ancestral to SF2 was much higher than for other lineages, with some sites having a ratio well above one. Positive selective pressure appears to have driven the divergence of SF2 from SF1 and SF3.

Nontargeted metabolome analysis by use of Fourier Transform Ion Cyclotron Mass Spectrometry.

Advanced functional genomic tools now allow the parallel and high-throughput analyses of gene and protein expression. Although this information is crucial to our understanding of gene function, it offers insufficient insight into phenotypic changes associated with metabolism. Here we introduce a high-capacity Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS)-based method, capable of nontargeted metabolic analysis and suitable for rapid screening of similarities and dissimilarities in large collections of biological samples (e.g., plant mutant populations). Separation of the metabolites was achieved solely by ultra-high mass resolution; Identification of the putative metabolite or class of metabolites to which it belongs was achieved by determining the elemental composition of the metabolite based upon the accurate mass determination; and relative quantitation was achieved by comparing the absolute intensities of each mass using internal calibration. Crude plant extracts were introduced via direct (continuous flow) injection and ionized by either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in both positive or negative ionization modes. We first analyzed four consecutive stages of strawberry fruit development and identified changes in the levels of a large range of masses corresponding to known fruit metabolites. The data also revealed novel information on the metabolic transition from immature to ripe fruit. In another set of experiments, the method was used to track changes in metabolic profiles of tobacco flowers overexpressing a strawberry MYB transcription factor and altered in petal color. Only nine masses appeared different between transgenic and control plants, among which was the mass corresponding to cyanidin-3-rhamnoglucoside, the main flower pigment. The results demonstrate the feasibility and utility of the FTMS approach for a nontargeted and rapid metabolic "fingerprinting," which will greatly speed up current efforts to study the metabolome and derive gene function in any biological system.

Muriel Wheldale Onslow (1880–1932): pioneer plant biochemist

The British biochemist Muriel Wheldale Onslow was one of only two genetics researchers in the first decade of the 20th century who performed plant breeding experiments and also investigated the corresponding chemistry of flower pigments of the plants (the other being Erwin Bauer in Germany)1. J.B.S. Haldane used her research to conclude that genes controlled the formation of large molecules, such as pigment molecules2.

Flower pigment composition of Crocus species and cultivars used for a chemotaxonomic investigation

A survey of floral anthocyanins and other flavonoids by analytical high-performance liquid chromatography (HPLC) was performed among 70 species and subspecies, 43 cultivars and six artificial hybrids of Crocus and the results were compared with taxonomical delimitations established by Mathew (The Crocus. B.T. Batsford Ltd, London, 1982). Nine anthocyanins were detected. The Crocus species and cultivars were placed into seven chemotypes according to their contents of 3,7- di - O -, 3,5- di - O -glucosides or 3- O -rutinosides of delphinidin and petunidin and to the presence of 3,7- di - O -malonyl-glucosides of petunidin and malvidin and delphinidin 3- O -glucoside-5- O -malonylglucoside. These malonated anthocyanins have only been found in Crocus and may be characteristic for this genus. The same 18 flavonoids were detected in every taxon. However, quantitative differences were noted and four chemotypes of Crocus were defined by their major contents of flavonoids. Six of the flavonoids appear to be unique for Crocus. The anthocyanin/flavonoid patterns of some of the taxa provide a valuable supplement to the taxonomy based on morphological and cytological patterns. Most chemotypes were represented in several series but the chemical data were useful in distinguishing different species. For all series except Series h the chemical data were very similar for all subspecies or accessions within a species, and chemotypes within a series were more similar than between series. However for six species, the analyses suggest that they should be further investigated using other methods, to evaluate their relations to other series.

Role of Photoreceptor- and Sugar -mediated Reactions in Light Dependent Anthocyanin Production in Lily and Stock Flowers

Low light intensity causes poor flower pigmentation possibly by two mechanisms : 1) the reaction mediated by photoreceptors located in the petal, 2) the reaction mediated by the sugar supply from leaves or stems. We investigated which mechanism is more important in the pigmentation of the oriental hybrid lily, 'Acapulco' and stock, 'Pigmy Rose'. Shading flowers by cheesecloth (PPFD<17μmol·m-2·sec-1) or by aluminum foil (0μmol·m-2·sec-1) reduced anthocyanin concentration in both species, suggesting that anthocyanin production is a photoreceptor-mediated reaction. When whole plants were shaded by cheesecloth (<17μmol·m-2·sec-1) in lily, anthocyanin concentration became lower than that of flower-shaded plants. This treatment reduced total sugar concentration from 3% to 1.6%, suggesting that limited sugar supply caused poor anthocyanin production in lily. In contrast, in stock, whole plant shading did not reduce anthocyanin concentration as compared with flower-shaded plants, suggesting that the amount of available sugar is not a limiting factor for anthocyanin production. Although this treatment reduced total sugar concentration from 4.7% to 3.7%, it was still higher than that of the control lily plants. When detached florets of stock were placed on sucrose solutions, the anthocyanin concentration declined as hexose concentration in the petal decreased, especially under 2%. These results indicate that, although soluble sugars in petals affected anthocyanin production, their high concentration prevented fading of flower color, even under low light conditions in stock.

Phenetics in Tree Peony Species from China by Flower Pigment Cluster Analysis

section Moutan DC. (fifteen accessions) from China. As the pigment markers, five anthocyanins together with three flavone and three flavonol aglycones were used. Principal component analysis (PCA) provided five eigenvectors from nineteen pigment patterns, in which peonidin and cyanidin as well as the types of glycosides greatly influenced the first factor, Z1. The Euclidean distances of standardized values obtained by eigenvector matrices with five factors (Z1 to Z5) produced a dendrogram in Ward's minimum variance cluster analysis, which showed good agreement with a classification based on the morphological characteristics of two subsections Vaginatae F.C. Stern and Delavayanae F.C. Stern. The results obtained by PCA and clustering qualify a chemical classification of section Moutan using secondary metabolites, specifically flower pigments, as taxonomic markers.

Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-3′,5′-hydroxylase: specific switching of the pathway in petunia

Flavonoid-3′,5′-hydroxylase (F3′5′H) is the key enzyme in the synthesis of 3′,5′-hydroxylated anthocyanins, which are generally required for the expression of blue or purple flower color. It has been predicted that the introduction of this enzyme into a plant species that lacks it would enable the production of blue or purple flowers by altering the anthocyanin composition. We present here the results of the genetic engineering of petunia flower color, pigmentation patterns and anthocyanin composition with sense or antisense constructs of the F3 ′ 5 ′ H gene under the control of the CaMV 35S promoter. When sense constructs were introduced into pink flower varieties that are deficient in the enzyme, transgenic plants showed flower color changes from pink to magenta along with changes in anthocyanin composition. Some transgenic plants showed novel pigmentation patterns, e.g. a star-shaped pattern. When sense constructs were introduced into blue flower petunia varieties, the flower color of the transgenic plants changed from deep blue to pale blue or even pale pink. Pigment composition analysis of the transgenic plants suggested that the F3 ′ 5 ′ H transgene not only created or inhibited the biosynthetic pathway to 3′,5′-hydroxylated anthocyanins but switched the pathway to 3′,5′-hydroxylated or 3′-hydroxylated anthocyanins.

SEPARATION OF ACYLATED ANTHOCYANIN PIGMENTS BY HIGH SPEED CCC

The reddish pigments of red onions (Allium cepa) and flowers of the tulip cultivar Los Angelos, including fragile anthocyanins with aliphatic acylation, were separated in semi-preparative scale by high speed countercurrent chromatography, HSCCC. The lower, polar phase of the biphasic solvent, t-butyl-methylether:n-butanol: acetonitrile:water (2:2:1:5 v/v) acidified with 0.01% trifluoroacetic acid, was used as mobile phase. All the five major anthocyanins in red onion, which contained the same aglycone (cyanidin), were separated from a 750 mg anthocyanin-enriched isolate. Relative to cyanidin 3-glucoside, analogous anthocyanins with additional acyl group(s) (malonyl) and sugar (glucosyl) entailed higher and lower elution volume, respectively. The major pigments in a 430 mg anthocyanin-enriched sample from flowers of the tulip “Los Angelos” were determined to be the 3-O-(6″-rhamnosylglucosides) of delphinidin, cyaniding, and pelargonidin, and the 3-O-[6″-(2‴-acetylrhamnosyl)glucosides] of cyanidin and pelargonidin. Acetylated anthocyanins were eluted after their non-acetylated analogues. Within each of the two groups, acetylated and non-acetylated, individual anthocyanins were separated according to their number of hydroxy-groups on the aglycone B-rings; highest number entailed lowest elution volume. All the acylated anthocyanins in the anthocyaninenriched isolates of both red onions and tulip “Los Angelos” were nearly baseline separated.

The role of nectar production, flower pigments and odour in the pollination of four species of Passiflora (Passifloraceae) in south-eastern Brazil

The role of nectar production, flower pigments and odour in the pollination of four species of Passiflora (Passifloraceae) in south-eastern Brazil

The role of nectar production, flower pigments and odour in the pollination of four species of Passiflora (Passifloraceae) in south-eastern Brazil

The pollination biology of four species of passionflower was studied in south-eastern Brazil, specifically the importance of chemical features of floral nectar, pigments and odours. All species required pollinators to produce fruits P. alata was pollinated by bees, P. speciosa by hummingbirds, and P. galbana and P. mucronata by bats. Pollinators consumed nectar as a food source. The activity of vertebrate pollinators reflected resource availability: they foraged when large amounts of nectar were available and when quantitative resource predictability was greater. The nectar of the vertebrate-pollinated species was richer in cholesterol and phospholipids, and had a potassium–sodium ratio higher than 1.0. For all species, the light absorption of flowers was paralleled by the pollinators» visual spectral sensitivity. This first report on Passiflora floral volatile compounds showed that there was a greater chemical class diversity among the species pollinated by animals with an acute olfactory sense, such as bees and bats. Benzenoid alcohols were the most represented compounds. The fragrances contained compounds that occur in other plant species and in the exocrine secretions of bees. This study shows a strong association between pollinators and the attracting and rewarding features of flowers.

Flower colours and pigments in hybrid tuberose ( Polianthes)

The flower pigment constitutions of nine hybrids with pink, reddish purple, purple, orange and yellow flowers obtained from the crossings and back-crossings using Polianthes tuberosa ‘Single’ and ‘Double’ and Polianthes howardii were analysed. Among the parents and hybrids, two white flowered cultivars of P. tuberosa had neither carotenoids nor anthocyanins, one hybrid had only carotenoids, four hybrids had only anthocyanins and an additional other four hybrids along with P. howardii had both carotenoids and anthocyanins in their petals. The main anthocyanidin in the petals of anthocyanin containing flowers is cyanidin with which some hybrids also contained delphinidin. Inducing of the anthocyanins and carotenoids from P. howardii into P. tuberosa can contribute to the extension of the diversity of flower colours in further tuberose breeding.