Flavonoid chemistry ofBlennospermatinae, a trans-Pacific disjunct subtribe ofSenecioneae (Asteraceae)

The flavonoid profiles of seven species ofAbrotanella and one species ofIschnea have been shown to be based upon kaempferol 3- and quercetin 3-O-glycosides and a delphinidin glycoside. Glucosides, glucuronides, arabinosides, diglucosides, and rutinosides of the flavonols were identified. The profile ofIschnea consisted solely of quercetin 3-O-glucoside and 3-O-arabinoside whereas the profiles of theAbrotanella species were more varied. Although infraspecific variation was not investigated in this study, the flavonoid chemistry of the two genera is in accordance with the flavonoid variation described for other members ofSenecioneae which are primarily flavonol producers. Based on the known phylogeny and biogeography, the flavonoid distribution from the perspective of long-distance dispersals across the Pacific is discussed. Such events should lead to genetic bottle-neck situations and depauperate flavonoid profiles. A summary of current flavonoid knowledge in theSenecioneae is supplied.     

Comparative phytochemistry of eleven species of Vigna (Fabaceae)

A survey of the biochemical constituents of 11 species of Vigna indicates the absence of the non-protein amino acid canavanine in their seeds, and absence of proanthocyanidin (polyphenol) in their leaves. Proanthocyanidin was found in the seeds of all, except Vigna subterranea. The constitutive leaf flavonoids of four genotypes of the pantropic V. subterranea were also studied and compared with those from three other cultivated species. The flavonoid kaempferol seems to be most prevalent as it was found in all of the four cultivated species and genotypes. The glycoside kaempferol-3-O-rutinoside was found present in the four genotypes of V. subterranea and other cultivated Vigna species. However, the flavonoid kaempferol-3-O-glucoside-7-rhamnoside is restricted to V. subterranea. This study questions the inclusion of V. subterranea in the genus Vigna on account of absence of seed proanthocyanidin and restricted accumulation of kaempferol-3-O-glucoside-7-rhamnoside in the leaves.     

Flavonoid genes of pear (<Emphasis Type="Italic">Pyrus communis</Emphasis>)

Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3β-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.     

Activation of flavonoid biosynthesis by solar radiation in bilberry (<Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> L.) leaves

The effect of solar radiation on flavonoid biosynthesis was studied in bilberry (Vaccinium myrtillus L.) leaves. Expression of flavonoid pathway genes of bilberry was studied in the upper leaves of bilberry, exposed to direct sunlight, in the shaded leaves growing lower in the same plants and in fruits. Bilberry-specific digoxigenin–dUTP-labeled cDNA fragments of five genes from the general phenylpropanoid pathway coding phenylalanine ammonia-lyase and from the flavonoid pathway coding chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase were used as probes in gene expression analysis. Anthocyanins, catechins, proanthocyanidins, flavonols and hydroxycinnamic acids from the leaves and fruits were identified and quantified using high-performance liquid chromatography combined with a diode array detector. An increase in the expression of the studied flavonoid pathway genes was observed in leaves growing under direct sun exposure. Also, the concentrations of anthocyanins, catechins, flavonols and hydroxycinnamic acids were higher in the leaves exposed to direct sunlight. However, the concentration of polymeric procyanidins was lower in sun-exposed leaves, whereas that of prodelphinidins was slightly increased. The results give further support for the protective role of flavonoids and hydroxy cinnamic acids against high solar radiation in plants. Also, the roles of different flavonoid compounds as a defense against stress caused by sun exposure is discussed.Abbreviations ANS Anthocyanidin synthase - CHS Chalcone synthase - DFR Dihydroflavonol 4-reductase - F3H Flavanone 3-hydroxylase - GPD Glyceraldehyde-3-phosphate dehydrogenase - PAL Phenylalanine ammonia-lyase     

Flavonoid chemistry ofWeigela (Caprifoliaceae) in Korea

Fifteen flavonoids were isolated from flowers and leaves of four species ofWeigela [W. florida (Bunge) A. DC.,W. praecox (Lemoine) Bailey,W. hortensis (Sieb. et Zucc.) K. Koch, andW. subsessilis (Nakai) Bailey] of Korea and one species (W. coraeensis Thunb.) of Japan. The flavonoid data indicated the presence of two distinct chemical groups: the “yellow flower” type producing flavonols and the “red flower” type producing flavonols and flavones. Two cyanidin 3-O-glycosides (glucoside and glucose-xylose) also occurred in all examined taxa. In the floral color-changing species,W. subsessilis, only quercetin glycosides predominated in floral tissue at first, decreasing in number and quantity with time. Instead, cyanidin 3-O-glycosides became present predominantly in flower color changing tissue from yellow to mauve.Weigela florida produced apigenin and luteolin glycosides, along with cyanidin 3-O-glycosides, which were also found inW. subsessilis. Within a relatively limited number of individuals (five),W. hortensis was unique in its production of all flavonols, flavones, and anthocyanins, although two individuals lacked flavone compounds but possessed all flavonols and anthocyanins. In effect, the putative hybrid,W. hortensis of Korea showed additive profiles of the parental marker compounds ofW. subsessilis andW. florida. Pollinator (andrenid bees) non-discrimination betweenWeigela flower-color morphs leading to non-assortive mating was a common, which indicated no breeding barrier among species. This flavonoid study indicated that species of both sections,Weigela andCalysphyrum appeared in each chemical grouping and it was obvious that the arrangement based on flavonoids cut across the sectional treatment of Hara. Floral tissues may be directly involved in the evolutionary strategy of pollination mechanisms and hence, their inherent flavonoids may no longer support taxonomic relationships. The presence of flavone glycosides inWeigela would support that tribe Dievilleae have a closer affinity to tribe Lonicereae within the Family Caprifoliaceae.     

Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin,proanthocyanidin, and flavonol levels during bilberry fruit development

The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.     

Flavonoid C- and O-glycosides from the Mongolian medicinal plant Dianthus versicolor Fisch

Eighteen flavonoids were identified from an aqueous extract of the aerial parts of Dianthus versicolor, a plant used in traditional Mongolian medicine against liver diseases. The flavonoid C- and O-glycosides isoorientin-7-O-rutinoside, isoorientin-7-O-rhamnosyl-galactoside, isovitexin-7-O-rutinoside, isovitexin-7-O-rhamnosyl-galactoside, isoscoparin-7-O-rutinoside, isoscoparin-7-O-rhamnosyl-galactoside, isoscoparin-7-O-galactoside, and isoorientin-7-O-galactoside were isolated and structurally elucidated. Their structures were established on the basis of extensive spectroscopic techniques including LC–UV–DAD, LC–MSⁿ, LC–HRMS, 1D and 2D NMR spectroscopy, and by GC–MS analysis after hydrolysis. Flavonoids with such a high glycosylation pattern are rare within the genus Dianthus. Furthermore, isovitexin-7-O-glucoside (saponarin), isovitexin-2″-O-rhamnoside, apigenin-6-glucoside (isovitexin), luteolin-7-O-glucoside, apigenin-7-O-glucoside, as well as the aglycons luteolin, apigenin, chrysoeriol, diosmetin, and acacetin were identified by TLC and LC–DAD–MSⁿ in comparison to reference substances or literature data. The NMR data of seven structures have not been reported in the literature to date.     

A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning,biochemical properties and developmentally regulated expression

Two flavonoid glucosyltransferases, UDP-glucose:flavonoid 3-O-glucosyltransferase (3-GT) and UDP-glucose: anthocyanin 5-O-glucosyltransferase (5-GT), are responsible for the glucosylation of anthocyani(di)ns to produce stable molecules in the anthocyanin biosynthetic pathway. The cDNAs encoding 3-GT and 5-GT were isolated from Petunia hybrida by hybridization screening with heterologous probes. The cDNA clones of 3-GT, PGT8, and 5-GT, PH1, encode putative polypeptides of 448 and 468 amino acids, respectively. A phylogenetic tree based on amino acid sequences of the family of glycosyltransferases from various plants shows that PGT8 belongs to the 3-GT subfamily and PH1 belongs to the 5-GT subfamily. The function of isolated cDNAs was identified by the catalytic activities for 3-GT and 5-GT exhibited by the recombinant proteins produced in yeast. The recombinant PGT8 protein could convert not only anthocyanidins but also flavonols into the corresponding 3-O-glucosides. In contrast, the recombinant PH1 protein exhibited a strict substrate specificity towards anthocyanidin 3-acylrutinoside, comparing with other 5-GTs from Perilla frutescens and Verbena hybrida, which showed broad substrate specificities towards several anthocyanidin 3-glucosides. The mRNA expression of both 3-GT and 5-GT increased in the early developmental stages of P. hybrida flower, reaching the maximum at the stage before flower opening. Southern blotting analysis of genomic DNA indicates that both 3-GT and 5-GT genes exist in two copies in P. hybrida, respectively. The results are discussed in relation to the molecular evolution of flavonoid glycosyltransferases.     

Pleiotropic changes in Arabidopsis <Emphasis Type="Italic">f5h</Emphasis> and <Emphasis Type="Italic">sct</Emphasis> mutants revealed by large-scale gene expression and metabolite analysis

Hydrocinnamic acid esters, lignin, flavonoids, glucosinolates, and salicylic acid protect plants against UV exposure, oxidative stress, diseases, and herbivores. Through the phenylpropanoid pathway, certain Brassicaceae family members, including Arabidopsis thaliana and Brassica napus, accumulate large amounts of the anti-nutritive sinapoylcholine (sinapine) in the seed. We successfully down-regulated activities of key enzymes in the pathway including F5H and SCT and achieved reduction of sinapine and lignin in B. napus seeds. Despite this success, it was unclear how multiple agronomic traits were affected in the transgenic plants. Here, we report altered large-scale gene expression of new alleles of f5h and sct mutants of A. thaliana and resultant accumulation of sinapoylglucose, disinapoylglucose, quercetin-3-O-rhamnoside, salicylic acid glucoside, and total indolyl glucosinolates in the two mutants. Expression of several flowering genes was altered in these mutants when grown under drought and NaCl treatments. Furthermore, both mutants were more susceptible to fungal infection than the wild type. Microarray experiments identified distinctive spatial and temporal expression patterns of gene clusters involved in silique/seed developmental processes and metabolite biosynthesis in these mutants. Taken together, these findings suggest that both f5h and sct mutants exhibit major differences in accumulation of diverse metabolites in the seed and profound changes in global large-scale gene expression, resulting in differential pleiotropic responses to environmental cues.     

Flavonoids of Heterogaura (Onagraceae)

Heterogaura is a monotypic genus of the tribe Onagreae of the Onagraceae. It is endemic to south western Oregon and California. Four flavonol glycosides, kaempferol 3-O-rhamnoside, quercetin 3-O-glucoside, quercetin 3-O-rhamnoglucoside and myricetin 3-O-glucoside, were found to occur in methanolic leaf extracts of each of the populations sampled. The presence of only flavonols is consistent with flavonoid analyses from other genera of the Onagreae, including Clarkia, the closest relative of Heterogaura.     

Flavonoids in the leaves of twenty-eight polygonaceous plants

Flavonoids in the leaves of twenty-eight species belonging to the Polygonaceae were studied. Thirty-three kinds of flavonoids were isolated, and eighteen kinds were obtained as crystals. Quercetin glycosides were commonly found in the family. In the quercetin glycosides, 3-O-rhamnoside was most frequently found: 3-O-glucuronide is also distributed widely. Myricetin glycosides were rare. Methylated flavonols were found in some species of the sectionsEchinocaulon andPersicaria. Eleven kinds ofC-glycosylflavones were found in the present survey, andC-glycosylflavones were distributed in all species of the genusRheum and in almost all species of the section Tiniaria.Rumex Acetosella andPolygonum suffultum are exceptional, the former contains flavone glycoside and the latterC-glycosylflavones only, as main components.     

An analysis of flavonoid compounds in leaves of Japonolirion (Petrosaviaceae)

Japonolirion, comprising Japonolirion osense Nakai, which occurs on serpentinite at two widely separated localities in Japan, has been considered as an isolated taxon, but more recently has been proved by molecular evidence to be a sister group to an achlorophyllous, mycoheterotrophic genus, Petrosavia. In an effort to research possible characters linking these groups, we analyzed the flavonoid compounds obtained from leaves of Japonolirion using UV spectra, mass spectrometry and ¹H and ¹³C nuclear magnetic resonance, and acid hydrolysis of the original glycosides as well as direct thin layer chromatography and high performance liquid chromatography comparisons with authentic specimens. As a result, we identified seven flavonoids, of which two were major components identified as 6-C-glucosylquercetin 3-O-glucoside and isoorientin. The remaining five were minor components identified as 6-C-glucosylkaempferol 3-O-glucoside, quercetin 3-O-glucoside, quercetin 3-O-arabinoside, vicenin-2 and orientin. Both 6-C-glucosylquercetin 3-O-glucoside and 6-C-glucosylkaempferol 3-O-glucoside were recorded for the first time in nature. Because of their restricted occurrence in angiosperms, both C-glycosylflavonols and 3-O-glycosides of C-glycosylflavonols may be significant chemical markers for assessing relationships of J. osense.     

The flavonoids of Stenosiphon (Onagraceae)

Stenosiphon linifolius is a monotypic genus of the tribe Onagreae of the Onagraceae. The species is widespread in, but restricted to, the Great Plains of the United States. Three flavonol glycosides, kaempferol 3-O-rhamnoside, quercetin 3-O-rhamnoside and myricetin 3-O-rhamnoside, were found to occur in methanolic extracts of Stenosiphon leaves. Similar compounds are found in the leaves of such related genera as Oenothera and Gaura, but in the latter genera, additional flavonols exhibiting greater substitutional variation also are found.     

Analysis of flavonoids and characterization of theOsFNS gene involved in flavone biosynthesis in Rice

The major flavonoids in rice leaves were analyzed via LC-MS/MS after their total flavonoid extracts were hydrolyzed. The most abundant flavones were apigenin, luteolin, and tricetin. Of these, tricetin was methylated at its 3′ and 5′-hydroxyl group to form tricin, which was probablyO-glycosylated. Both 3′-O-methylated luteolin and luteolin were found in theC-glycosylated form while apigenin wasC-glycosylated. We also cloned and characterizedOsFNS, which catalyzes the reaction from flavanone (naringenin) to flavone (apigenin). Analysis of the reaction product with recombinant OsFNS showed that it indeed converts naringenin to apigenin.     

The formation of flavonoids in cell suspension cultures ofPrunus x yedoensis matsum

Two lines of the red and pale yellow cell suspension cultures, prepared fromPrunus x yedoensis Matsum. callus induced by Murashige and Skoog's (1962) basal medium supplemented with 2, 4-dichlorophenoxyacetic acid (2, 4-D, 1.0 mg/l), kinetin (0.1 mg/l) and sucrose (30 g/l), were maintained on Schenk and Hildebrandt medium as modified by Mitchell and Gildow (1975). The red cell suspension culture produced cyanidin 3-monoglucoside, 5, 4′-dihydroxy-7-methoxyisoflavone 4′-glucoside (prunetrin), isoquercitrin, catechin, epicatechin, and procyanidin B-1, B-2, B-3 and B-4, while the pale yellow cells produced only a small amount of catechin and epicatechin as main flavonoids. These flavonoid compounds found in the red cell culture were present also in maturePrunus leaves. Maximum growth and maximum amount of total phenol and proanthocyanidin (procyanidins) were obtained with 0.3 mg/l of both 2,4-D and kinetin. Maximum concentration of anthocyanin was also obtained with 0.3 mg/l 2, 4-D regardless of kinetin concentration. Accumulation of proanthocyanidin was markedly stimulated by low concentrations of phosphate, which reduced growth by about half, and also by high concentrations of inorganic nitrogen. Production of both anthocyanin and proanthocyanidin was reduced by lowered nitrogen levels. Cell growth and production of all phenolics were inhibited when ammonium ion replaced nitrate in the medium.     

Synthesis,release, and transmission of alfalfa signals to rhizobial symbionts

In addition to the flavonoids exuded by many legumes as signals to their rhizobial symbionts, alfalfa (Medicago sativa L.) releases two betaines, trigonelline and stachydrine, that induce nodulation (nod) genes inRhizobium meliloti. Experiments with¹⁴C-phenylalanine in the presence and absence of phenylalanine ammonia-lyase inhibitors show that exudation of flavonoidnod-gene inducers from alfalfa roots is linked closely to their concurrent synthesis. In contrast, flavonoid and betainenod-gene inducers are already present on mature seeds before they are released during germination. Alfalfa seeds and roots release structurally differentnod-gene-inducing signals in the absence of rhizobia. WhenR. meliloti is added to roots, medicarpin, a classical isoflavonoid phytoalexin normally elicited by pathogens, and anod-gene-inducing compound, formononetin-7-O-(6-O-malonylglycoside), are exuded. Carbon flow through the phenylpropanoid pathway and into the flavonoid pathway via chalcone synthase is controlled by complexcis-acting sequences andtrans-acting factors which are not completely understood. Even less information is available on molecular regulation of the two other biosynthetic pathways that produce trigonelline and stachydrine. Presumably the three separate pathways for producingnod-gene inducers in some way protect the plant against fluctuations in the production or transmission of the two classes of signals. Factors influencing transmission of alfalfanod-gene inducers through soil are poorly defined, but solubility differences between hydrophobic flavonoids and hydrophilic betaines suggest that the diffusional traits of these molecules are not similar. Knowledge derived from studies of how legumes regulate rhizobial symbionts with natural plant products offers a basis for defining new fundamental concepts of rhizosphere ecology.     

山樱花品种间花色差异的代谢组学研究

山樱花是世界著名的观花类植物，花色是其最重要的观赏特征。为探究影响山樱花品种间花色差异的代谢通路及关键代谢产物变化，该文利用LC-MS/MS技术对白色、绿色和粉色的山樱花品种进行花青素靶向代谢组学比较分析。结果表明：(1)共检测到42种花青素物质，主要包含矮牵牛素、飞燕草素、黄酮类化合物、锦葵色素、芍药花素、矢车菊素、天竺葵素和原花青素8种物质。(2)差异代谢花青素25种，包括11种下调、14种上调，其中有7种花青素在粉色花瓣中显著富集。(3)KEGG通路注释发现差异代谢物在花青素生物合成通路中显著富集，结合聚类结果发现矮牵牛素-3-O-葡萄糖苷是山樱花品种间花色差异产生的关键代谢物。该研究揭示了山樱花花色差异的代谢机理，为后续山樱花花色分子调控机制研究提供了一定的理论依据，也为新品种花色改良和选育提供了一定的科学参考。     

A new alpinumisoflavone derivative from Genista pichisermolliana

The aerial parts of Genista pichisermolliana Valsecchi (Fabaceae), an endemic plant of Sardinia, were extracted in Soxhlet apparatus and purified by several chromatographic methods. The new compound alpinumisoflavone 4′-O-glucopyranoside (6) was isolated together with nineteen flavonoids, p-coumaric methylester and d-pinitol, while no alkaloids were detected. All the chemical structures were elucidated by spectroscopic analysis. Since flavonoids represent the main constituents of this plant, the total flavonoid content was determined according to the Italian Pharmacopoeia IX Ed. method.