Flavonoid distribution in Pyracantha coccinea plants at different growth phases

Flavonoid composition during the ontogenetic cycle was examined in Pyracantha coccinea. The flavonoid profiles of plants at different ages showed marked differences in aerial and hypogeal parts. In the vegetative phase there are flavonoids (flavanones, flavones, and flavonols) only in the aerial parts and they appear gradually during the plant life. These secondary metabolites are detectable in the roots exclusively in the reproductive phase.     

A DIGE-based quantitative proteomic analysis of grape berry flesh development and ripening reveals key events in sugar and organic acid metabolism

Grapevine (Vitis vinifera L.) is an economically important fruit crop. Quality-determining grape components, such as sugars, acids, flavours, anthocyanins, tannins, etc., are accumulated during the different grape berry development stages. Thus, correlating the proteomic profiles with the biochemical and physiological changes occurring in grape is of paramount importance to advance the understanding of the berry development and ripening processes. Here, the developmental analysis of V. vinifera cv. Muscat Hamburg berries is reported at protein level, from fruit set to full ripening. A top-down proteomic approach based on differential in-gel electrophoresis (DIGE) followed by tandem mass spectrometry led to identification and quantification of 156 and 61 differentially expressed proteins in green and ripening phases, respectively. Two key points in development, with respect to changes in protein level, were detected: end of green development and beginning of ripening. The profiles of carbohydrate metabolism enzymes were consistent with a net conversion of sucrose to malate during green development. Pyrophosphate-dependent phosphofructokinase is likely to play a key role to allow an unrestricted carbon flow. The well-known change of imported sucrose fate at the beginning of ripening from accumulation of organic acid (malate) to hexoses (glucose and fructose) was well correlated with a switch in abundance between sucrose synthase and soluble acid invertase. The role of the identified proteins is discussed in relation to their biological function, grape berry development, and to quality traits. Another DIGE experiment comparing fully ripe berries from two vintages showed very few spots changing, thus indicating that protein changes detected throughout development are specific.     

Phenolic compounds in berries and flowers of a natural hybrid between bilberry and lingonberry (Vaccinium × intermedium Ruthe)

Hybridization between species plays an important role in the evolution of secondary metabolites and in the formation of combinations of existing secondary metabolites in plants. We have investigated the content of phenolic compounds in berries and flowers of Vaccinium × intermedium Ruthe, which is a rare natural hybrid between bilberry (Vaccinium myrtillus L.) and lingonberry (Vaccinium vitis-idaea L.). The berries and flowers of the hybrid showed characteristics inherited from both parent species in the distribution and contents of phenolic compounds. Bilberry is known as one of the richest sources of anthocyanins and to have a profile of 15 major forms combining cyanidin, delphinidin, petunidin, peonidin and malvidin with galactose, glucose and arabinose. Lingonberry contains only cyanidin glycosides. Hybrid berries contained all bilberry anthocyanins with pronounced cyanidin content. With regard to proanthocyanidins and flavonol glycosides, the hybrid inherited diverse profiles combining those of both parental species. The distribution of hydroxycinnamic acids was quite uniform in all studied berries. Of the identified compounds, 30 were detected in lingonberry, 46 in bilberry, 53 in hybrid berries and 38 in hybrid flowers. Hence, compared with the parent species, hybrid berries possess a more diverse profile of phenolic compounds and, therefore, can offer interesting material for breeding purposes.     

小苍兰花色色素成分及稳定性分析

以小苍兰(Freesia refracta)16个不同花色品种及后代为试验材料,对花瓣色素用特征显色反应和紫外-可见光谱扫描,分析其色素的成分和花色素苷的稳定性.结果表明,小苍兰花色的色素属于类黄酮化合物,含黄酮和花色素苷类物质,可能含有异黄酮,不含黄酮醇、二氢黄酮、二氢黄酮醇、查耳酮和橙酮,其中黄色系品种及后代还含有类胡萝卜素.避光下小苍兰花色素苷的稳定性要强于光照;温度对花瓣色素的稳定性有一定的影响.     

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.     

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.     

Metabolomics reveals organ-specific metabolic rearrangements during early tomato seedling development

Tomato seedlings (Solanum lycopersicum cv. MoneyMaker), grown under strictly controlled conditions, have been used to study alterations occurring in secondary metabolite biosynthetic pathways following developmental and environmental perturbations. Robustness and reproducibility of the system were confirmed using detailed statistical analyses of the metabolome. LCMS profiling was applied using whole germinated seeds as well as cotyledons, hypocotyls and roots from 3 to 9 days old seedlings to generate relative levels of 433 metabolites, of which 62 were annotated. Initial focus was given to the polyphenol pathway and several additional mass signals have been putatively annotated using high mass resolution fragmentation. Clear organ and developmental stage—specific differences were observed. Seeds accumulated saponin-like compounds; roots accumulated mainly alkaloids; cotyledons contained mainly glycosylated flavonols and; hypocotyls contained mainly anthocyanins. For each organ, the developmental changes in metabolite profiles were described by using linear mixed models. Across three independent experiments, 85 % of the metabolites showed similar developmental trends. This tomato seedling system has given us valuable additional insights into the complexity of seedling secondary metabolism. How metabolic profiles reflect an interplay between depletion of stored molecules and de novo synthesis and how the overall picture for this important crop plant contrasts to e.g. Arabidopsis are emphasised.     

Flavone synthase II (CYP93B16) from soybean (Glycine max L.)

Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4′-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNS II was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.     

Differences in metabolite profiles between blood matrices,ages, and sexes among Caucasian individuals and their inter-individual variations

Endobiotic metabolites are associated with biological processes in the body and therefore may serve as biomarkers for disease states or therapeutic efficacy and toxicity. However, information is limited regarding how differences between blood matrices, patient backgrounds, and sample handling affect human metabolite profiles. Our objective was to obtain metabolite profiles from Caucasian individuals, based on different matrices (plasma and serum), subject backgrounds (male/female and young/old), and storage conditions (2 or 10 freeze–thaw cycles). In total, 297 metabolites were detected by LC/MS and GC/MS, and more than 75 % of them were highly represented in all sample groups. The multivariate discriminant analysis (OPLS-DA as a model) singled out the matrix type as the most important variable influencing global metabolic profiles; that is, more than 100 metabolites were significantly different based on the matrix type. The influence of subject backgrounds on global metabolic profiles was consistent between plasma and serum. Age-associated differences were more predominant in females than males, whereas gender-associated differences were more prevalent in young subjects than old individuals were. The relative standard deviation of metabolite levels in subjects with the same background ranked from 0.1 to 1.5. Moreover, the changes of metabolite levels caused by freeze–thaw cycles were limited, and the effect was more prominent in plasma than serum. These data demonstrate the impact of matrix, age, gender, and freeze–thaw cycles on the metabolite profiles and reveal metabolites affected by these factors. Thus, our results provide would useful fundamental information for exploring and qualifying biomarkers for clinical applications.     

Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation

Flavones are important copigments found in the flowers of many higher plants and play a variety of roles in plant adaptation to stress. In Medicago species, flavones also act as signal molecules during symbiotic interaction with the diazotropic bacterium Sinorhizobium meliloti. They are the most potent nod gene inducers found in root exudates. However, flavone synthase II (FNS II), the key enzyme responsible for flavone biosynthesis, has not been characterized in Medicago species. We cloned two FNS II genes from Medicago truncatula using known FNS II sequences from other species and named them MtFNSII-1 and MtFNSII-2. Functional assays in yeast (Saccharomyces cerevisiae) suggested that the catalytic mechanisms of both cytochrome P450 monooxygenases were similar to the other known legume FNS II from licorice (Glycyrrhiza echinata). MtFNSII converted flavanones to 2-hydroxyflavanones instead of flavones whereas FNS II from the nonlegume Gerbera hybrida, converted flavanones to flavones directly. The two MtFNSII genes had distinct tissue-specific expression patterns. MtFNSII-1 was highly expressed in roots and seeds whereas MtFNSII-2 was highly expressed in flowers and siliques. In addition, MtFNSII-2 was inducible by S. meliloti and methyl jasmonate treatment, whereas MtFNSII-1 was not. Histochemical staining of transgenic hairy roots carrying the promoter-reporter constructs indicated that the MtFNSII-2 induction was tissue specific, mostly localized to vascular tissues and root hairs. RNA interference-mediated suppression of MtFNSII genes resulted in flavone depleted roots and led to significantly reduced nodulation when inoculated with S. meliloti. Our results provide genetic evidence supporting that flavones are important for nodulation in M. truncatula.     

The Structure and Distribution of the Flavonoids in Plants

6 -C₃-C₆ skeleton, have been found in plants, and are divided into several classes, i.e., anthocyanins, flavones, flavonols, flavanones, dihydroflavonols, chalcones, aurones, flavan and proanthocyanidins, isoflavonoids, biflavonoids, etc. In this review, the chemical structures of the reported flavonoid classes are introduced and their distribution in nature are described. Additionally, some recent chemotaxonomical examples using the flavonoids are also given. Received 1 June 2000/ Accepted in revised form 1 July 2000     

Metabolomics reveals abundant flavonoids in edible insect Antheraea pernyi

The natural flavonoids in foods of plant origin have been well-characterized due to their beneficial biological properties. However, the information regarding the flavonoid compounds in edible insects remains severely limited. In the present study, we used a metabolomics approach to identify the flavonoid compounds in the Chinese oak silkworm, Antheraea pernyi Guérin-Méneville (Lepidoptera: Saturniidae), an traditional edible insect. Our study identified over 200 flavonoid metabolites in the larval midgut of A. pernyi with LC-ESI-MS/MS system. These flavonoid metabolites come from eight subclasses, including flavones (1 0 3), flavonols (34), flavonoids (28), flavanones (20), polyphenols (19), isoflavones (9), anthocyanins (9), and proanthocyanidins (4). The relative content of the flavones is the most abundant, with a value of 36.74% of the total. The top five flavonoid components in A. pernyi are hyperoside, isoquercitroside, tricin 7-O-hexoside, hesperetin 5-O-glucoside and protocatechuic acid, accounting for 51.17% of the total flavonoids. Hyperoside is the most abundant flavonoid compound (18.07% of the total) in A. pernyi. Our findings indicated targeted metabolomics is a useful approach to identify flavonoids in edible insects which contain abundant flavonoids than we already knew.     

Correlated accumulation of anthocyanins and rosmarinic acid in mechanically stressed red cell suspensions of basil (Ocimum basilicum)

A red basil cell line (T2b) rich in rosmarinic acid (RA) was selected for the stable production of anthocyanins (ACs) in the dark. Cell suspension cultures were subjected to mechanical stress through increased agitation (switch from 90 to 150 rpm) to determine the relationship between AC and RA accumulation. Cell extracts were analyzed by HPLC and LC-MS, and the resulting data were processed with multivariate statistical analysis. MS and MS/MS spectra facilitated the putative annotation of several complex cyanidin-based ACs, which were esterified with coumaric acid and, in some cases, also with malonic acid. It was also possible to identify various RA-related molecules, some caffeic and coumaric acid derivatives and some flavanones. Mechanical stress increased the total AC and RA contents, but reduced biomass accumulation. Many metabolites were induced by mechanical stress, including RA and some of its derivatives, most ACs, hydroxycinnamic acids and flavonoids, whereas the abundance of some RA dimers was reduced. Although AC and RA share a common early biosynthetic pathway (from phenylalanine to 4-coumaroyl-CoA) and could have similar or overlapping functions providing antioxidant activity against stress-generated reactive oxygen species, there appeared to be no competition between their individual pathways.     

FLAVONOID EVOLUTION IN ROBINSONIA (COMPOSITAE) OF THE JUAN FERNANDEZ ISLANDS

Leaf flavonoids were isolated and identified from 54 populations representing all seven species of Robinsonia, a genus of dioecious rosette trees endemic to the Juan Fernandez Islands. Fourteen compounds were detected consisting of flavonols, flavones, flavanones and dihydroflavonols. The distribution of these compounds in Robinsonia largely corresponds to specific and sectional limits based on morphological data. The morphologically similar species, R. gayana and R. thurifera, have identical flavonol profiles (derivatives of quercetin). Likewise, the closely related R. evenia and R. masafuerae are unique in the genus by possessing flavones. The inclusion of Rhetinodendron (i.e., R. berteroi) in Robinsonia is supported by its strong flavonoid similarity with species in two other sections of the genus. The morphologically diverse section Eleutherolepis exhibits the greatest flavonoid variation of any section, and only here are found flavones, flavanones and dihydroflavonols. The direction of flavonoid evolution in Robinsonia is hypothesized to be from fewer to more classes of compounds. Biosynthetic considerations suggest that this gain in compounds is due both to a gain of an additional enzymatic step and to the sequestering of precursors. This interpretation of direction of flavonoid evolution is in agreement with several lines of evidence including the flavonoid chemistry of the hypothesized outgroup (i.e., species of Senecio on mainland Chile), the ages of the two islands, and morphological trends.     

Evidence for a putative flavonoid translocator similar to mammalian bilitranslocase in grape berries (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) during ripening

During maturation, Vitis vinifera berries accumulate a large amount of several anthocyanins in the epidermal tissue, whereas their precursors and intermediates are ubiquitously synthesized within the fruit. Up to date, several mechanisms of flavonoid transport at subcellular level have been hypothesized, but it is not possible to identify a general model applicable in every plant tissue and organ. Recently, a putative anthocyanin carrier, homologue to mammalian bilitranslocase (BTL) (TC 2.A.65.1.1), was found in Dianthus caryophyllus petal microsomes. In the present paper, an immunohistochemical and immunochemical analysis, using an antibody raised against a BTL epitope, evidences the expression and function of such a transporter in V. vinifera berries (cv. Merlot). Specific localisations of the putative carrier within berry tissues together with expression changes during different developmental stages are shown. Water stress induces an increase in protein expression in both skin and pulp samples. A bromosulfalein (BSP) uptake activity, inhibitable by the BTL antibody, is detected in berry mesocarp microsomes, with K (m) = 2.39 microM BSP and V (max) = 0.29 micromol BSP min(-1) mg(-1) protein. This BSP uptake is also competitively inhibited by quercetin (K (i) = 4 microM). A putative role for this carrier is discussed in relation to the membrane transport of secondary metabolites.