Flower colour and cytochromes P450

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.     

Anatomical and biochemical studies of bicolored flower development in Muscari latifolium

The inflorescence of the broad-leafed grape hyacinth, Muscari latifolium, shows an interesting, two-tone appearance with the upper flowers being pale blue and the lower ones purple. To elucidate the mechanism of the differential color development, anatomical research was carried out and a cytological study of the colored protoplasts in which the shapes of the cells accumulating anthocyanin were observed by scanning electron microscopy. Next, vacuolar pH was recorded using a pH meter with a micro combination pH electrode, and the sap’s metal-ion content was measured by inductively coupled plasma mass spectrometry. The anthocyanin and co-pigment composition was determined by high-performance liquid chromatography (HPLC). Chemical analyses reveal that the difference in metal-ion content of the two parts was not great. The vacuolar pHs of the upper and lower flowers were 5.91 and 5.84, respectively, with the difference being nonsignificant. HPLC results indicate that the dihydroflavonol and flavonol contents are also very similar in the two sorts of flower. However, the upper flowers contained only delphinidin, whereas the lower flowers also contained cyanidin. The total anthocyanin content in the lower flowers was 4.36 mg g^?1, which is approximately seven times higher than in the upper flowers, while the delphinidin content is four times higher. Quantitative real-time PCR analysis established that the two-tone flower was a result of different expressions of the F3′5′H, F3′H and DFR genes, and these lead to different amounts of anthocyanin.     

Chromatographic and spectral characterization of 3′-glycosylation in anthocyanidins

The caffeyl ester of cyanidin 3,7,3′-triglucoside was isolated from red petals of garden cineraria, Senecio cruentus. Chromatographic and spectral characteristics of the anthocyanin as well as the products of deacylation and partial hydrolysis are described. The results with this pigment and a similar one based on delphinidin show that anthocyanins having a sugar residue at the 3′(or 5′)-position are characterized by the position of the visible max and by the high values for E₄₄₀/E_{vis max} and E_{UV max}/E_{vis max} when compared with other glycosides.     

The identification of flavonoids and the expression of genes of anthocyanin biosynthesis in the chrysanthemum flowers

In order to provide additional information on the coloration of chrysanthemum flowers, the flavonoid composition and the expression of six structural genes involved in anthocyanin pathway in the ray florets of a pink flowering (cv. H5) and two white flowering (cvs. Keikai and Jinba) Chrysanthemum grandiflorum cultivars were examined. HPLCDAD/ESI-MSⁿ analysis showed that cyanidin 3-O-(6″-O-malonylglucoside) and cyanidin 3-O-(3″,6″-O-dimalonylglucoside) were the two major flavonoids presented in H5, while white flowering cultivars contained flavones instead of anthocyanins. Nine flavone derivatives were detected in the three cultivars, the amount of each flavone varied upon cultivars, and seven of these were identified as luteolin 7-O-arabinosylglucuronide, apigenin 7-O-glucoside, luteolin 7-O-malonylglucoside, apigenin 7-O-malonylglucoside, chrysoeriol 7-O-malonylglucoside, acacetin 7-O-rutinoside and acacetin 7-O-malonylglucoside. The two white flowering cultivars showed similar total flavonoid content, which was about two fold higher than that in H5. A high expression of the genes encoding dihydroflavonol 4-reductase and 3-O-glucosyltransferase was detected only in H5 but not in Keikai or Jinba. Chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, and flavonoid 3′-hydroxylase were expressed in all flowers, suggesting that the lack of anthocyanin in white flowering cultivars cannot be due to any blockage of their expression.     

铁筷子花瓣中花青素苷成分及含量对其呈色的影响北大核心CSCD

该研究以7个品种铁筷子(Helleborus thibetanus Franch.)为试验材料,借助目视测色、RHSCC比色卡、色差仪进行花色表型的测定,采用高效液相色谱法-光电二极管阵列检测方法(HPLC-DAD)及高效液相色谱-电喷雾离子化-质谱联用技术(HPLC-ESI-MS)测定分析铁筷子花瓣中花青素苷成分及含量,以探究不同品种铁筷子的花色与花青素苷成分及含量之间的关系。结果显示:(1)紫色系品种花瓣的a*值最高b*值最低,黄色系品种花瓣的b*值最高a*值最低,不同品种的铁筷子花色越深L*值越低。(2)从5个有花青素苷积累的铁筷子品种中检测出11种花青素苷成分,分别为6种矢车菊素苷,4种飞燕草素苷,1种矮牵牛素苷;供试的铁筷子材料中红色系2个品种的花青素苷含量最高,紫色系品种次之;矢车菊素苷与飞燕草素苷为影响铁筷子花瓣呈色的主要色素物质。(3)不同种类的花青素和修饰基团的差异,导致铁筷子花瓣呈现不同的色彩,含有多种酰基化修饰的飞燕草素苷使铁筷子花色蓝移进而使花色加深。(4)相关分析表明,铁筷子花瓣的L*值与a*值呈显著负相关关系,与b*值呈显著的正相关关系;L*值与总花青素苷含量呈显著负相关关系,且随着花青素苷含量的累积a*值增加,花色红移。研究表明,花青素苷的成分及含量是导致铁筷子花瓣呈现不同颜色的主要原因,矢车菊素苷和飞燕草素苷的互作以及酰基化的修饰使铁筷子呈现不同程度的紫色,花青素苷的不同累积量影响了花瓣颜色的明暗变化,从而使铁筷子花瓣颜色丰富。     

Differential expression of anthocyanin biosynthetic genes in relation to anthocyanin accumulation in the pericarp of Litchi chinensis Sonn

Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m⁻² among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT was found significantly correlated with the pericarp anthocyanin concentration (r = 0.84). These results suggest that UFGT played a predominant role in the anthocyanin accumulation in litchi as well as pericarp coloration of a given cultivar.     

Cyanidin glycosides in flowers of genus Corydalis (Fumariaceae)

Nine taxa of Corydalis were surveyed for their floral anthocyanins. Five cyanidin glycosides: cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside, cyanidin 3-(2^G-xylosylrutinoside) and cyanidin 3-(2^G-xylosylrutinoside)-7-glucoside were isolated from these taxa and identified by chemical and spectroscopic techniques. A novel anthocyanin was found in the flowers of Corydalis elata and Corydalis flexuosa cultivars, and identified to be cyanidin 3-(2^G-xylosylrutinoside)-7-glucoside. Two anthocyanins, cyanidin 3-sambubioside and cyanidin 3-(2^G-xylosylrutinoside), were also found for the first time in Corydalis flowers. Furthermore, the major anthocyanin constituent of the flowers was cyanidin 3-sambubioside in the outer petals of Corydalis ambigua and Corydalis lineariloba, and cyanidin 3-rutinoside in those of Corydalis decumbens, Corydalis curvicalcarata and Corydalis speciosa. Similarly, Corydalis incisa contained cyanidin 3-(2^G-xylosylrutinoside), and C. flexuosa ‘China Blue’ and ‘Blue Panda’, and C. elata contained the most complex structural pigment, cyanidin 3-(2^G-xylosylrutinoside)-7-glucoside, as their dominant anthocyanin in their outer petals. According to the results of anthocyanin analyses, these nine plants were classified into four groups: groups A (three taxa), B (two taxa), C (one taxa) and D (three taxa). On the other hand, the anthocyanin constituent of their inner petals was composed of cyanidin 3-rutinoside as only one dominant anthocyanin.     

Cyanidin 3-p-coumaroylglucoside in Camellia species and cultivars

The major anthocyanin of red flowers of Camellia hiemalis, C. japonica and C. sasanqua was determined to be cyanidin 3-O-β-d-(6-O-p-coumaroylglucoside) by fast atom bombardment mass spectrometry and NMR spectroscopy. It was identical with the pigment, hyacinthin, of the bulb scales of Hyacinthus orientalis. This pigment and cyanidin 3-glucoside are widely distributed in the flowers of Camellia japonica and many Camellia cultivars.     

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.     

Electronically Monitored Labial Dabbing and Stylet ‘Probing’ Behaviors of Brown Marmorated Stink Bug,Halyomorpha halys,in Simulated Environments

Brown marmorated stink bug, Halyomorpha halys (Stål), (Hemiptera: Pentatomidae) is an invasive polyphagous agricultural and urban nuisance pest of Asian origin that is becoming widespread in North America and Europe. Despite the economic importance of pentatomid pests worldwide, their feeding behavior is poorly understood. Electronically monitored insect feeding (EMIF) technology is a useful tool in studies of feeding behavior of Hemiptera. Here we examined H. halys feeding behavior using an EMIF system designed for high throughput studies in environmental chambers. Our objectives were to quantify feeding activity by monitoring proboscis contacts with green beans, including labial dabbing and stylet penetration of the beans, which we collectively define as ‘probes’. We examined frequency and duration of ‘probes’ in field-collected H. halys over 48 hours and we determined how environmental conditions could affect diel and seasonal periodicity of ‘probing’ activity. We found differences in ‘probing’ activity between months when the assays were conducted. These differences in activity may have reflected different environmental conditions, and they also coincide with what is known about the phenology of H. halys. While a substantial number of ‘probes’ occurred during scotophase, including some of the longest mean ‘probe’ durations, activity was either lower or similar to ‘probing’ activity levels during photophase on average. We found that temperature had a significant impact on H. halys ‘probing’ behavior and may influence periodicity of activity. Our data suggest that the minimal temperature at which ‘probing’ of H. halys occurs is between 3.5 and 6.1°C (95% CI), and that ‘probing’ does not occur at temperatures above 26.5 to 29.6°C (95% CI). We estimated that the optimal temperature for ‘probing’ is between 16 and 17°C.     

Synthesis and monitored selection of nucleotide surrogates for binding T:A base pairs in homopurine-homopyrimidine DNA triple helices

A total of 16 oligodeoxyribonucleotides of general sequence 5′-TCTTCTZTCTTTCT-3′, where Z denotes an N-acyl-N-(2-hydroxyethyl)glycine residue, were prepared via solid phase synthesis. The ability of these oligonucleotides to form triplexes with the duplex 5′-AGAAGATAGAAAGA-HEG-TCTTTCTATCTTCT-3′, where HEG is a hexaethylene glycol linker, was tested. In these triplexes, an ‘interrupting’ T:A base pair faces the Z residue in the third strand. Among the acyl moieties of Z tested, an anthraquinone carboxylic acid residue linked via a glycinyl group gave the most stable triplex, whose UV melting point was 8.4°C higher than that of the triplex with 5′-TCTTCTGTCTTTCT-3′ as the third strand. The results from exploratory nuclease selection experiments suggest that a combinatorial search for strands capable of recognizing mixed sequences by triple helix formation is feasible.     

Flavonoids from the flowers of Adenium obesum (Forssk.) Roem. & Schult., Mandevilla sanderi (Hemsl.) Woodson,and Nerium oleander L. (Apocynaceae)

Twenty-two ornamental flowers from different Adenium obesum, Mandevilla sanderi, and Nerium oleander cultivars/seedlings were analyzed for the presence of anthocyanins, flavonols, and chlorogenic acid using nuclear magnetic resonance (NMR) and mass spectrometry (MS). Cyanidin 3-O-[6-O-(rhamnosyl)-galactoside] and cyanidin 3-O-(galactoside) were identified as the major and minor anthocyanins, respectively, in three A. obesum seedlings that had red and red-purple flowers.Cyanidin 3-O-[2-O-(xylosyl)-galactoside] was identified as the major anthocyanin, whereas cyanidin 3-O-[6-O-(rhamnosyl)-galactoside] and cyanidin 3-O-(galactoside) were identified as the minor anthocyanins in 8 M. sanderi cultivars that had red and red-purple flowers. Cyanidin 3-O-[6-O-(rhamnosyl)-galactoside] and cyanidin 3-O-(galactoside) were identified as the major anthocyanins, whereas cyanidin 3-O-[2-O-(xylosyl)-galactoside] was identified as the minor anthocyanin in 8 N. oleander cultivars with red and red-purple flowers. Low levels of anthocyanins were detected in the N. oleander and M. sanderi cultivars that had white flowers, and there were no anthocyanins detected in the N. oleander cultivars with yellow flowers. Chlorogenic acid and four flavonols, quercetin 3-O-[6-O-(rhamnosyl)-galactoside], quercetin 3-O-[6-O-(rhamnosyl)-glucoside], kaempferol 3-O-(galactoside), and kaempferol 3-O-[6-O-(rhamnosyl)-galactoside], were identified in the flowers from all 22 cultivars/seedlings investigated.     

Origin of Chrysanthemum cultivars — Evidence from nuclear low-copy LFY gene sequences

The chrysanthemums (Chrysanthemum × morifolium Ramat.) are a well-known group of traditional ornamental flowers in China and have been cultivated all over the world. Yet the origin of chrysanthemum cultivars has been highly debated. In this study we employ the nuclear low-copy LFY gene to study the evolutionary history of chrysanthemum cultivars. The structure of the LFY gene in all Chrysanthemum species examined is highly conserved with three exons and two introns. The length of the LFY gene in Chrysanthemum varied from 2, 887 to 3, 348 bp. The two introns exhibited high levels of variation in length and sequence composition at the intraspecific and interspecific levels. Phylogenetic analysis of the whole LFY sequences of Chrysanthemum resulted in topologies that contained three major clades. The LFY sequences from the same cultivars are present in two or three clades, supporting that hybridization and allopolyploidy were important mechanisms in the origins of different chrysanthemums. Our results suggest that different cultivars had different ancestors. Chrysanthemum indicum, C. zawadskii and C. nankingense were likely the direct ancestors of most chrysanthemum cultivars examined. Chrysanthemum vestitum is a putative ancestor for some cultivars, and may have indirectly involved in the development of the chrysanthemum cultivars. Sequences of the LFY gene are informative to shed insights into the origin of chrysanthemum cultivars and show great potential as a phylogenetic marker to decipher the phylogeny of Chrysanthemum and its close relatives.