Flavonoid diversification in different leaf compartments of Primula auricula (Primulaceae)

Populations of Primula auricula L. subsp. auricula from Austrian Alps were studied for flavonoid composition of both farinose exudates and tissue of leaves. The leaf exudate yielded Primula-type flavones, such as unsubstituted flavone and its derivatives, while tissue flavonoids largely consisted of flavonol 3-O-glycosides, based upon kaempferol (3, 4) and isorhamnetin (5–7). Kaempferol 3-O-(2″-O-β-xylopyranosyl-[6″-O-β-xylopyranosyl]-β-glucopyranoside) (3) and isorhamnetin 3-O-(2″-O-β-xylopyranosyl-[6″-O-β-xylopyranosyl]-β-glucopyranoside) (6) are newly reported as natural compounds. Remarkably, two Primula type flavones were also detected in tissues, namely 3′-hydroxyflavone 3′-O-β-glucoside (1) and 3′,4′-dihydroxyflavone 4′-O-β-glucoside (2), of which (1) is reported here for the first time as natural product. All structures were unambiguously identified by NMR and MS data. Earlier reports on the occurrence of 7,2′-dihydroxyflavone 7-O-glucoside (macrophylloside) in this species could not be confirmed. This structure was now shown to correspond to 3′,4′-dihydroxyflavone 4′-O-glucoside (2) by comparison of NMR data. Observed exudate variations might be specific for geographically separated populations. The structural diversification between tissue and exudate flavonoids is assumed to be indicative for different ecological roles in planta.     

Variation in flavonoid pattern in leaves and flowers of Primula veris of different origin and impact of UV-B

Flavonoids can serve as chemotaxonomic markers and play an important role in protection against ultraviolet (UV) radiation. Primula veris originating from two natural field sites in Albania and one cultivar from Austria were used to investigate whether flavonoid pattern may differ between populations and to determine their response to UV. Plants were grown in a common environment and shortly before flowering transferred in two greenhouses with 80% and 4% UV-B transmission, respectively. After two weeks, young leaves and open flowers were harvested and flavonoids analyzed by high performance liquid chromatography. The flavonoid profiles of leaves and flowers were highly distinct for each population, with certain flavonoids occurring only in plants of particular field sites. These flavonoids may be useful biomarkers to identify the origin of plant material. The differences in UV-treatment at that stage had no effect on the total flavonoid contents of both leaves and flowers. However, individual flavonoids of both leaves and flowers responded sensitively to UV, suggesting that they may be involved in protection against UV.     

Interpopulational variation in the flavonoid composition of Cistus ladanifer L. exudate

A study was made in different populations of Cistus ladanifer of the quantitative and qualitative variation of the exudate of the leaves and stems, including the exudate's aglycone flavonoid content. Two groups of populations were established according to different climate conditions, and the results showed the existence of interpopulational variability both quantitatively and qualitatively. The populations subject to the greatest thermal stress and moisture stress (Group I) synthesized more exudate and total flavonoids. The amounts of the different flavonoids synthesized also differed between the two groups of populations: Group I secreted more 3-O-methylkaempferol, 3,4′-di-O-methylkaempferol and 3,7-di-O-methylkaempferol and Group II more 4′-O-methylapigenin. Quantifying the proportion of each apigenin and kaempferol relative to their totals, it was observed that the synthesis of position 7 methylated flavonoids are enhanced in Group I, and position 4′ methylated flavonoids in Group II. The indication is that the climatic conditions to which a population of C. ladanifer is subject may lead to diversification in the flavonoid composition of the leaf and stem exudate, with the implication that flavonoids could have various ecological functions in this species.     

DNA Barcoding Evaluation and Its Taxonomic Implications in the Species-Rich Genus Primula L. in China

The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA) and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.     

Exudate flavonoid aglycones in the alpine species of Achillea sect. Ptarmica: Chemosystematics of A. moschata and related species (Compositae-Anthemideae)

In completion of our studies on Achillea exudate flavonoids, 11 alpine species of Achillea sect. Ptarmica were analyzed for their aglycone profiles. The study focuses on species commonly associated with A. moschata. The major flavonoid constituents found in exudates of most taxa were 6-hydroxyflavonol 3,6,4'-trimethyl ethers, except in A. ageratifolia and its subspecies, which are characterized by the accumulation of the 3,6,7-trimethoxy and 6-hydroxyflavones. Infraspecific variation was particularly high in A. abrotanoides and A. moschata. Results are discussed in relation to published data for related species and within the context of evolutionary aspects in the genus Achillea. The 3,6,4'-trimethoxy substitution is regarded as a basic chemical trend within the genus Achillea. Geographical and ecological aspects are briefly addressed, and a summary on known exudate aglycone composition of species from all sections of Achillea is included.     

Ethylene Response Factor (ERF) genes modulate plant root exudate composition and the attraction of plant parasitic nematodes

Plant root exudates are compositionally diverse, plastic and adaptive. Ethylene signalling influences the attraction of plant parasitic nematodes, presumably through the modulation of root exudate composition. Understanding this pathway could lead to new sources of crop parasite resistance. Here we used Virus-Induced Gene Silencing to knock down the expression of two Ethylene Response Factor (ERF) genes, ERF-E2 and ERF-E3, in tomato. Root exudates were significantly more attractive to the PPNs Meloidogyne incognita and Globodera pallida following knockdown of ERF-E2, which had no impact on the attraction of Meloidogyne javanica. Knockdown of ERF-E3 had no impact on the attraction of Meloidogyne or Globodera spp. Gas Chromatography Mass Spectrometry analysis revealed major changes in root exudate composition relative to controls. However, these changes did not alter the attraction of rhizosphere microbes Bacillus subtilis or Agrobacterium tumefaciens. This study further supports the potential of engineering plant root exudate for parasite control, through the modulation of plant genes.     

Vegetable Exudates as Food for Callithrix spp. (Callitrichidae): Exploratory Patterns

Marmosets of the genus Callithrix are specialized in the consumption of tree exudates to obtain essential nutritional resource by boring holes into bark with teeth. However, marmoset preferences for particular tree species, location, type, and other suitable factors that aid in exudate acquisition need further research. In the current study, the intensity of exudate use from Anadenanthera peregrina var. peregrina trees by hybrid marmosets Callithrix spp. groups was studied in five forest fragments in Viçosa, in the state of Minas, Brazil. Thirty-nine A. peregrina var. peregrina trees were examined and 8,765 active and non-active holes were analyzed. The trunk of A. peregrina var. peregrina had a lower number of holes than the canopy: 11% were found on the trunk and 89% were found on the canopy. The upper canopy was the preferred area by Callithrix spp. for obtaining exudates. The intensity of tree exploitation by marmosets showed a moderate-to-weak correlation with diameter at breast height (DBH) and total tree height. The overall results indicate that Anadenanthera peregrina var. peregrina provides food resources for hybrid marmosets (Callithrix spp.) and these animals prefer to explore this resource on the apical parts of the plant, where the thickness, location, and age of the branches are the main features involved in the acquisition of exudates.     

Increasing flavonoid concentrations in root exudates enhance associations between arbuscular mycorrhizal fungi and an invasive plant

Many invasive plants have enhanced mutualistic arbuscular mycorrhizal (AM) fungal associations, however, mechanisms underlying differences in AM fungal associations between introduced and native populations of invasive plants have not been explored. Here we test the hypothesis that variation in root exudate chemicals in invasive populations affects AM fungal colonization and then impacts plant performance. We examined flavonoids (quercetin and quercitrin) in root exudates of native and introduced populations of the invasive plant Triadica sebifera and tested their effects on AM fungi and plant performance. We found that plants from introduced populations had higher concentrations of quercetin in root exudates, greater AM fungal colonization and higher biomass. Applying root exudates more strongly increased AM fungal colonization of target plants and AM fungal spore germination when exudate donors were from introduced populations. The role of root exudate chemicals was further confirmed by decreased AM fungal colonization when activated charcoal was added into soil. Moreover, addition of quercetin into soil increased AM fungal colonization, indicating quercetin might be a key chemical signal stimulating AM fungal associations. Together these results suggest genetic differences in root exudate flavonoids play an important role in enhancing AM fungal associations and invasive plants’ performance, thus considering root exudate chemicals is critical to unveiling mechanisms governing shifting plant-soil microbe interactions during plant invasions.Subject terms: Population dynamics, Community ecology, Plant ecology     

UGT84F9 is the major flavonoid UDP-glucuronosyltransferase in Medicago truncatula

Mammalian phase II metabolism of dietary plant flavonoid compounds generally involves substitution with glucuronic acid. In contrast, flavonoids mainly exist as glucose conjugates in plants, and few plant UDP-glucuronosyltransferase enzymes have been identified to date. In the model legume Medicago truncatula, the major flavonoid compounds in the aerial parts of the plant are glucuronides of the flavones apigenin and luteolin. Here we show that the M. truncatula glycosyltransferase UGT84F9 is a bi-functional glucosyl/glucuronosyl transferase in vitro, with activity against a wide range of flavonoid acceptor molecules including flavones. However, analysis of metabolite profiles in leaves and roots of M. truncatula ugt84f9 loss of function mutants revealed that the enzyme is essential for formation of flavonoid glucuronides, but not most flavonoid glucosides, in planta. We discuss the use of plant UGATs for the semi-synthesis of flavonoid phase II metabolites for clinical studies.

UGT84F9 is a bifunctional glucuronosyltransferase/glucosyltransferase that is necessary for the glucuronidation of a wide range of flavonoid natural products in Medicago truncatula.     

Flavonoids in seed and root exudates of Lotus pedunculatus and their biotransformation by Mesorhizobium loti

Analysis of the flavonoid content of seed and root exudates of Lotus pedunculatus was undertaken using multiple coupled analytical techniques: capillary zone electrophoresis coupled to a UV spectral array detector (CZE-UV), high performance thin-layer chromatography with densitometry (HPTLC-UV) and gas chromatography with mass spectrometry (GC-MS). These procedures provided separation, identification and structural confirmation of the previously unidentified flavonoids in this plant's seed and root exudates and were particularly applicable to samples from a small seeded legume. Catechin, naringenin, kaempferol, quercetin aglycone and 3 different glycosides of quercetin were detected in seed exudate. Sterile root exudates contained catechin, naringenin and quercetin in addition to apigenin, kaempferol and other flavones and flavanones for which partial identifications were obtained. When sterile root exudate was incubated with Mesorhizobium loti , changes in its flavonoid content were detected. Analysis of bacterial cells after incubation revealed the presence of quercetin, kaempferol and one other flavone. The monocyclic aromatics protocatechuic acid and phloroglucinol were detected in both the incubated root exudate and its bacterial cells.     

Root hairs are the most important root trait for rhizosheath formation of barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)

Background and AimsRhizosheaths are defined as the soil adhering to the root system after it is extracted from the ground. Root hairs and mucilage (root exudates) are key root traits involved in rhizosheath formation, but to better understand the mechanisms involved their relative contributions should be distinguished.MethodsThe ability of three species [barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)] to form a rhizosheath in a sandy loam soil was compared with that of their root-hairless mutants [bald root barley (brb), maize root hairless 3 (rth3) and root hairless 1 (Ljrhl1)]. Root hair traits (length and density) of wild-type (WT) barley and maize were compared along with exudate adhesiveness of both barley and maize genotypes. Furthermore, root hair traits and exudate adhesiveness from different root types (axile versus lateral) were compared within the cereal species.Key ResultsPer unit root length, rhizosheath size diminished in the order of barley > L. japonicus > maize in WT plants. Root hairs significantly increased rhizosheath formation of all species (3.9-, 3.2- and 1.8-fold for barley, L. japonicus and maize, respectively) but there was no consistent genotypic effect on exudate adhesiveness in the cereals. While brb exudates were more and rth3 exudates were less adhesive than their respective WTs, maize rth3 bound more soil than barley brb. Although both maize genotypes produced significantly more adhesive exudate than the barley genotypes, root hair development of WT barley was more extensive than that of WT maize. Thus, the greater density of longer root hairs in WT barley bound more soil than WT maize. Root type did not seem to affect rhizosheath formation, unless these types differed in root length.ConclusionsWhen root hairs were present, greater root hair development better facilitated rhizosheath formation than root exudate adhesiveness. However, when root hairs were absent root exudate adhesiveness was a more dominant trait.     

Exudate flavonoids in some Gnaphalieae and Inuleae (Asteraceae)

Three members of the tribe Gnaphalieae and six members of the tribe Inuleae (Asteraceae) were analyzed for their exudate flavonoids. Whereas some species exhibit rather trivial flavonoids, others produce rare compounds. Spectral data of rare flavonoids are reported and their structural identification is discussed. 6-Oxygenation of flavonols is a common feature of two Inula species and Pulicaria sicula. By contrast, flavonoids with 8-oxygenation, but lacking 6-oxygenation, are common in two out of three Gnaphalieae species examined. In addition, B-ring deoxyflavonoids are abundantly present in the leaf exudates of Helichrysum italicum (Gnaphalieae). These distinctive features of the two Asteraceae tribes are in agreement with previous flavonoid surveys of these and related taxa.     

Flavonoids from chemotypes of the goldback fern, Pityrogramma triangularis

More than 20 flavonoid aglycones were identified from the frond exudate of various collections of Pityrogramma triangularis. Ten of them are novel natural products; most of the others are rare flavonoids. They are substituted at C-8; many are C-methylated. They mostly occur only as trace constituents in var. triangularis. Two compounds come from var. viscosa. One population of P. triangularis var. triangularis exhibits a unique flavonoid pattern not encountered before in any other chemotype studied.     

Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest,Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.     

Ethnobotanical and antibacterial study of Primula plants traditionally used in the indigenous communities of Western Himalaya,Pakistan

BackgroundThe upper belt of Azad Kashmir is a hilly, mountainous, and remote area where the indigenous communities mainly believe in traditional medicines for the treatment of different ailments. This study aimed to conserve scientifically and culturally important medicinal knowledge of Primula species in Azad Kashmir, Western Himalaya, Pakistan. The additional objective was to evaluate the antibacterial activity of these plants against pathogenic bacteria.MethodsThe ethnomedicinal data of Primula species was explored by conducting structured interviews with 40 informants of the study area, especially asking about the medicinal uses of Primula species. The indigenously used Primula species were further analyzed for their antibacterial activity against both gram-positive and gram-negative bacteria by using disc diffusion assay supplemented with a more robust minimum inhibitory concentration assay.ResultsEthnomedicinal data revealed that indigenous communities living in upper regions of Azad Kashmir use 5 Primula species for the treatment of various disorders. The highly cited disease category was ophthalmic disorders. P. denticulata and P. macrophylla were the most cited plant species with higher use reports such as 104 and 93, respectively. One or more extracts of different parts of Primula species showed a noteworthy antibacterial activity against one or more tested bacteria.ConclusionThis study provides novel information regarding several categories of traditional uses and antibacterial activity of Primula species in Azad Kashmir, Western Himalaya. The need for novel and more effective drugs derived from natural products is more important than ever, making future studies on herbal remedies both justified and urgently required.     

Flavonoids of Gardenia cramerii and G. fosbergii bud exudates

From the bud exudates of Gardenia cramerii and G. fosbergii, two species endemic to Sri Lanka, a new flavonoid with an unusual B-ring oxidation pattern, 5,5′-dihydroxy-6,7,2′,3′-tetramethoxyflavone, was characterized. Two other rare flavonoids, 5,3′,5′-trihydroxy-3,6,7,4′-tetramethoxyflavone and 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone were also isolated from both Gardenia species.     

Chemodiversity of exudate flavonoids in seven tribes of Cichorioideae and Asteroideae (Asteraceae)

Members of several genera of Asteraceae, belonging to the tribes Mutisieae, Cardueae, Lactuceae (all subfamily Cichorioideae), and of Astereae, Senecioneae, Helenieae and Heliantheae (all subfamily Asteroideae) have been analyzed for chemodiversity of their exudate flavonoid profiles. The majority of structures found were flavones and flavonols, sometimes with 6- and/or 8-substitution, and with a varying degree of oxidation and methylation. Flavanones were observed in exudates of some genera, and, in some cases, also flavonol- and flavone glycosides were detected. This was mostly the case when exudates were poor both in yield and chemical complexity. Structurally diverse profiles are found particularly within Astereae and Heliantheae. The tribes in the subfamily Cichorioideae exhibited less complex flavonoid profiles. Current results are compared to literature data, and botanical information is included on the studied taxa.     

Advances in the biotechnological glycosylation of valuable flavonoids

The natural flavonoids, especially their glycosides, are the most abundant polyphenols in foods and have diverse bioactivities. The biotransformation of flavonoid aglycones into their glycosides is vital in flavonoid biosynthesis. The main biological strategies that have been used to achieve flavonoid glycosylation in the laboratory involve metabolic pathway engineering and microbial biotransformation. In this review, we summarize the existing knowledge on the production and biotransformation of flavonoid glycosides using biotechnology, as well as the impact of glycosylation on flavonoid bioactivity. Uridine diphosphate glycosyltransferases play key roles in decorating flavonoids with sugars. Modern metabolic engineering and proteomic tools have been used in an integrated fashion to generate numerous structurally diverse flavonoid glycosides. In vitro, enzymatic glycosylation tends to preferentially generate flavonoid 3- and 7-O-glucosides; microorganisms typically convert flavonoids into their 7-O-glycosides and will produce 3-O-glycosides if supplied with flavonoid substrates having a hydroxyl group at the C-3 position. In general, O-glycosylation reduces flavonoid bioactivity. However, C-glycosylation can enhance some of the benefits of flavonoids on human health, including their antioxidant and anti-diabetic potential.     

Lipophilic exudates of Pteridaceae - chemistry and chemotaxonomy

A number of fern species, belonging to several genera of Pteridaceae, exhibit a more or less conspicuous farinose wax, which is mostly located on the lower leaf surface. Production of these waxes is often correlated with the presence of glandular trichomes. Particularly during the past two decades, a series of publications appeared on the chemical composition of these exudates. The major components were found to be flavonoids (chalcones, dihydrochalcones, flavanones, dihydroflavonols, flavones, flavonols), some of them with a complex substitution pattern, including esters and C-methyl derivatives, and even bisflavonoids. Diterpenoids and triterpenoids can also occur in such exudates. It is the purpose of this paper to survey the chemical composition of Pteridaceae exudates and their occurrence within the genera of the family. The chemotaxonomic significance of the flavonoid aglycones at the generic, specific and populational level is briefly discussed.