Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus

Microbial glycosyltransferases can convert many small lipophilic compounds such as phenolics, terpenoids, cyanohydrins and alkaloids into glycons using uridine-diphosphate-activated sugars. The main chemical functions of glycosylation processes are stabilization, detoxification and solubilization of the substrates. The gene encoding the UDP-glycosyltransferase from Bacillus cereus, BcGT-1, was cloned by PCR and sequenced. BcGT-1 was expressed in Escherichia coli BL21 (DE3) with a his-tag and purified using a His-tag affinity column. BcGT-1 could use apigenin, genistein, kaempferol, luteolin, naringenin and quercetin as substrates and gave two reaction products. The enzyme preferentially glycosylated at the 3-hydroxyl group, but it could transfer a glucose group onto the 7-hydroxyl group when the 3-hydroxyl group was not available. The reaction products made by biotransformation of flavonoids with E. coli expressing BcGT-1 are similar to those produced with the purified recombinant enzyme. Thus, this work provides a method that might be useful for the biosynthesis of flavonoid glucosides and for the glycosylation of related compounds.     

Engineering of flavonoid O-methyltransferase for a novel regioselectivity

An O-methyltransferase isolated from poplar, POMT7, was identified as a flavone 7-O-methyltransferase. In order to generate a mutant of POMT-7 having a novel regioselectivity, we conducted an error-prone polymerase chain reaction. More than 100 mutants were screened and one of the mutants (POMT-M1) Asp257Gly, methylated the 3-hydroxyl group of flavonols in addition to 7-hydrdoxyl group. The mutation changed asparagine residue at the position of 257 into glycine. The kinetic parameters showed that the wild type POMT7 was better activity toward kaempferol and quercetin than the POMT7-M1. Using E. coli transformant expressing POMT7-M1, 58 μM of 3, 7-O-dimethylquercetin and 70 μM of 3, 7-O-dimethylkaempferol from 100 μM of corresponding substrate were synthesized successfully.     

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.     

The flavonoid chemistry of the genusTrillium

Four flavonol glycosides (Fig.1) were isolated from the leaves ofTrillium tschonoskii Maxim. By means of UV, NMR, and mass spectral analyses, they were identified to be acetylated kaempferol 3-O-arabinosylgalactoside (TK-1), kaempferol 3-O-arabinosylgalactoside (TK-2), acetylated quercetin 3-O-arabinosylgalactoside (TQ-1) and quercetin 3-O-arabinosylgalactoside (TQ-2). High performance liquid chromatography (HPLC) profiles of 172 specimens ofT. tschonoskii collected from nine different places in Japan were grouped into three different types based on the flavonoid components: type I and type II containing TK-1 and TQ-1, and TK-2 and TQ-2, respectively, as main component, and type III containing all of four flavonol glycosides. Those results show that the intraspecific variation ofT. tschonoskii with different geographical distribution has not only been found by the analysis of karyotype, but also that of flavonoid components.     

Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae)

The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar range of non-acylated flavonoids.     

Acylated flavonoid tetraglycoside from Planchonia careya leaves

Phytochemical investigations of the aqueous extract of Planchonia careya leaves revealed two known flavonol glycosides, kaempferol 3-O-gentiobioside (1) and quercetin 3-O-glucoside (isoquercitrin) (2), and a novel acylated kaempferol tetraglycoside, kaempferol 3-O-[α-rhamnopyranosyl(1 → 3)-(2-O-p-coumaroyl)]-β-glucopyranoside, 7-O-[α-rhamnopyranosyl-(1 → 3)-(4-O-p-coumaroyl)]-α-rhamnopyranoside (3). Structural elucidation was achieved using UV, NMR, and mass spectrometry.     

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1α subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1α as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC₅₀ = 5.16 μM). The mechanism of this inhibition did not involve suppression of HIF-1α protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC₅₀ = 4.75 μM). Exposure of Huh7 cells to 10 μΜ kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 μM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.     

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.     

Study on Degradation of Flavonols in Mutants of PoppyPapaver somniferumL.

We studied flavonol-degrading activity of cell-free extracts from petals of the flower color and structure mutants. The relationship between degradation of flavonols (kaempferol, quercetin, and myricetin) and biosynthesis of anthocyanins has been revealed. The white-flower mutant proved to have the highest flavonol-degrading activity toward all substrates, particularly quercetin. The mutations inhibiting synthesis of pelargonidin, an anthocyanin, provide for synthesis of various amounts of cyanidin in the petals. The flavonol-degrading activity considerably increases proportionally to the content of cyanidin. A similar relationship has been revealed in the mutants synthesizing both cyanidin and pelargonidin. The plants accumulating considerable amounts of pelargonidin in their petals have accordingly higher flavonol-degrading activity and predominantly hydrolyze kaempferol. The plants forming additional pods in their flower (pistillody) have higher flavonol-degrading activity as compared to the anther-in-petal and doubleness mutants     

Variability of Flavonol Contents during Floral Morphogenesis in the Poppy Papaver somniferum L.

We studied the contents of flavonols (kaempferol and quercetin) in the meristem of vegetative and generative apices of the main plant shoot in floral Papaver somniferum L. mutants, as well as in the normal plants at successive stages of flower development. Five stages of flower development were distinguished. Flavonols (kaempferol and quercetin) were present in all flower organs at all stages of floral morphogenesis we studied. However, their contents and distribution in different organs and at different stages of flower development markedly varied. No significant differences were found in the contents of flavonols in the meristems of vegetative and generative apices of the main shoot in the lines of floral mutants, as well as between the lines with different amounts of vegetative phytomeres. In the plants with normal flower structure, the contents of flavonols (kaempferol + quercetin) sharply increased with the beginning of differentiation of flower organs, i.e. from stage 3, to reach a maximum in the open flower, when gametogenesis is terminated and fertilization takes place. The level of flavonol contents in the petals (upper part) and stamen was at a maximum at all stages of flower development, while that in the gynaecium was at a minimum. The kaempferol : quercetin ratio was shifted towards quercetin at successive stages of flower development, most significantly in the stamens. The involvement of flavonols in the regulation of floral morphogenesis at stages of flower organs differentiation and functioning is discussed.     

Variability of flavonol contents during floral morphogenesis in Papaver somniferum L

We studied the contents of flavonols (kaempferol and quercetin) in the meristem of vegetative and generative apices of the main plant shoot in floral Papaver somniferum mutants, as well as in the normal plants at successive stages of flower development. Five stages of flower development were distinguished. Flavonols (kaempferol and quercetin) were present in all flower organs at all stages of floral morphogenesis we studied. However, their contents and distribution in different organs and at different stages of flower development markedly varied. No significant differences were found in the contents of flavonols in the meristems of vegetative and generative apices of the main shoot in the lines of floral mutants, as well as between the lines with different amounts of vegetative phytomeres. In the plants with normal flower structure, the contents of flavonols (kaempferol + quercetin) sharply increased with the beginning of differentiation of flower organs, i.e. from stage 3, to reach a maximum in the open flower, when gametogenesis is terminated and fertilization takes place. The level of flavonol contents in the petals (upper part) and stamen was at a maximum at all stages of flower development, while that in the gynaecium was at a minimum. The kaempferol: quercetin ratio shifted towards quercetin at successive stages of flower development, most significantly in the stamens. The involvement of flavonols in the regulation of floral morphogenesis at stages of flower organs differentiation and functioning is discussed.     

Genomic organization of a UDP-glucosyltransferase gene determines differential accumulation of specific flavonoid glucosides in tepals

Glycosylation plays a major role in the chemical diversity of flavonoids. The wide diversity of the family-1 glycosyltransferase (UGT) impairs the determination of the biochemical function solely from its primary sequence. Here we combined differential expression and target metabolomic analysis in various Crocus species to identify a gene that is key in determining the flavonoid composition of Crocus species that belong to the Crocus series. UGT703B1 recognizes isorhamnetin and kaempferol as substrates in vitro. In addition, UGT703B1 expression was found to be highly correlated with the presence of kaempferol 7-O-biglucoside-3-O-β-glucoside and isorhamnetin-3,7-O-diglucoside. These flavonols were present in C. sativus and C. cartwrightianus albus, both from series Crocus but absent in Crocus species from the other series analyzed. Further, the presence of both flavonols was associated with the expression of UGT703B1, and this expression was correlated with the presence of the UGT703B1 coding gene, with the exception of C. cancellatus, whose genomic sequence was present but contained a shorter intronic sequence and promoter alterations, suggesting the presence of regulatory sequences in the deleted part of that intron and promoter important for UGT703B1 expression. Overall, the data obtained supports the involvement of UGT703B1 in the formation of specific kaempferol and isorhamnetin glucosides, while demonstrating that the integration of metabolomic and differential expression analysis is a versatile tool for understanding a multigene family of UGTs in Crocus.     

Effect of arsenic on flavonoid contents in Pteris species

Two arsenic (As) hyperaccumulators (Pteris multifida and Pteris vittata) and a non-hyperaccumulator (Pteris semipinnata) were exposed to different As concentrations under hydroponic conditions. Five flavonoids in these fern species were determined by high-performance liquid chromatography (HPLC). Flavonoid production in P. multifida and P. semipinnata was also studied in 0 and 20 mg As L⁻¹ treatments at different cultivation times. No significant differences were observed regarding the contents of quercetin, isoquercitrin and kaempferol in the fronds. The contents of rutin, quercetin, kaempferol and total flavonoids were also not significantly different in the roots of the three fern species under the same As treatment. However, significant differences were observed in contents of rutin, quercetin, hyperin, kaempferol and total flavonoids over time in the 20 mg As L⁻¹ treatment. In general, the changes in flavonoid contents in the As hyperaccumulators were not directly related to As accumulation.     

Study of neuroprotective function of Ginkgo biloba extract (EGb761) derived‐flavonoid monomers using a three‐dimensional stem cell‐derived neural model

An in vitro three‐dimensional (3D) cell culture system that can mimic organ and tissue structure and function in vivo will be of great benefit for drug discovery and toxicity testing. In this study, the neuroprotective properties of the three most prevalent flavonoid monomers extracted from EGb 761 (isorharmnetin, kaempferol, and quercetin) were investigated using the developed 3D stem cell‐derived neural co‐culture model. Rat neural stem cells were differentiated into co‐culture of both neurons and astrocytes at an equal ratio in the developed 3D model and standard two‐dimensional (2D) model using a two‐step differentiation protocol for 14 days. The level of neuroprotective effect offered by each flavonoid was found to be aligned with its effect as an antioxidant and its ability to inhibit Caspase‐3 activity in a dose‐dependent manner. Cell exposure to quercetin (100 µM) following oxidative insult provided the highest levels of neuroprotection in both 2D and 3D models, comparable with exposure to 100 µM of Vitamin E, whilst exposure to isorhamnetin and kaempferol provided a reduced level of neuroprotection in both 2D and 3D models. At lower dosages (10 µM flavonoid concentration), the 3D model was more representative of results previously reported in vivo. The co‐cultures of stem cell derived neurons and astrocytes in 3D hydrogel scaffolds as an in vitro neural model closely replicates in vivo results for routine neural drug toxicity and efficacy testing. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:735–744, 2016     

Enzymatic hydrolysis of green tea seed extract and its activity on 5alpha-reductase inhibition

Two kaempferol glycosides were isolated from green tea seed extract (GTSE). After conducting a structure analysis, these two compounds were identified as kaempferol-3-O-[2-O-beta-D-galactopyranosyl-6-O-alpha-L-rhamnopyranosyl]-beta-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-beta-D-xylopyranosyl-6-O-alpha-L-rhanmopyranosyl]-beta-D-glucopyranoside (compound 2). These two compounds were hydrolysed by o-glycolytic enzymes for the production of kaempferol. After performing several reactions, we found the optimum enzyme combination, a reaction with beta-galactosidase and hesperidinase. Finally, we produced kaempferol of above 95% purity. The 5alpha-reductase inhibition activities of GTSE hydrolysate (GTSE-H) containing kaempferol were evaluated by the contact cell-based metabolic method using a stable HEK 293 cell line. GTSE-H showed a good inhibition effect on HEK 293 cell lines both type 1 and type 2 on 5alpha-reductase. Especially, GTSE-H inhibited type 2 with kaempferol content dependency. The results indicate that the inhibition activity of hydrolysate on 5alpha-reductase type 2 increases in accordance with kaempferol content.     

Variations in flavonoid patterns within the genus Chondropetalum (restionaceae)

HPLC and chemical analyses of the flavonoids in culms of 11 Chondropetalum species divide the genus into two groups: seven, with glycosides of myricetin larycitin and syringetin; and four, with glycosides of kaempferol, quercetin, gossypetin, gossypetin 7-methyl ether and herbacetin 4′-methyl ether. This chemical dichotomy is correlated with anatomical differences and confirms the view that the genus requires taxonomic revision. HPLC measurements on those species with myricetin derivatives show that taxa with a qualitatively similar pattern of glycosides can be readily separated on quantitative grounds. Syringetin 3-arabinoside and a glycoside of herbacetin 4′-methyl ether are reported for the first time from the genus.     

Implications of long-distance flavonoid movement in Arabidopsis thaliana

Flavonoid synthesis is modulated by developmental and environmental signals that control the amounts and localization of the diverse flavonoids found in plants. Flavonoids are implicated in regulating a number of physiological processes including UV protection, fertilization, auxin transport, plant architecture, gravitropism and pathogenic and symbiotic interactions with other organisms. Recently we showed that flavonoids can move long distances in plants, which may facilitate these molecules reaching positions in the plant where these processes are regulated. The localised application of selective flavonoids to tt4 mutants such as naringenin, dihydrokaempferol and dihydroquercetin showed that they were taken up at the root tip, mid-root or cotyledons and travelled long distances via cell-to-cell movement to distal tissues and converted to quercetin and kaempferol. In contrast, kaempferol and quercetin do not move long distances. They were taken up only at the root tip and did not move from this position. Here we show the movement of endogenous flavonoids by using reciprocal grafting experiments between tt4 and wild-type seedlings. These results demonstrated that to understand the distribution of flavonoids in Arabidopsis, it is necessary to know where the flavonoid biosynthetic enzymes are made and to understand the mechanisms by which certain flavonoids move from their site of synthesis.Key words: flavonoid movement, reciprocal graft, quercetin, kaempferol, Arabidopsis thaliana, fluorescence, aglyconeFlavonoids are plant secondary metabolites made by the phenylpropanoid pathway. The central biosynthetic pathway is known and in Arabidopsis most of the enzymes in flavonoid synthesis are encoded by single copy genes.¹ The isolation of mutants with defects in the genes encoding these flavonoid biosynthetic enzymes has allowed researchers to understand the biochemical complexity of flavonoid synthesis and their biological roles. Flavonoid synthesis is more complex in other species, such as legumes, which produce a greater diversity of flavonoid molecules, and in which gene families encode the key enzymatic branch points of the pathway.^2,3The functions of flavonoids were demonstrated using genetic approaches that blocked flavonoid synthesis in Arabidopsis and other species. In Arabidopsis, flavonoids play important roles in UV protection⁴ and regulate auxin transport and dependent physiological processes, such as gravity responses,^5–7 and lateral root formation.⁸ In petunia, maize and tomato, pollen without flavonoids is infertile and this phenotype is reversed by flavonoid addition.^9–11 However, the enigma of why flavonoid-deficient Arabidopsis seedlings are fertile has not been resolved.¹² Flavonoids appear to interact with Multidrug resistance (MDR)/P-glycoproteins (PGP)/ABC-Type B proteins⁷ that transport auxin, regulate phosphatases and kinases, and may have regulatory roles as scavengers of reactive oxygen species (reviewed in ref. ¹³). These results are consistent with a diversity of important functions for flavonoids in plants that require careful control of flavonoid synthesis and localization.We have explored the possibility that flavonoid accumulation in specific locations is also modulated by movement of early intermediates of the flavonoid pathway. Long-distance movement of secondary metabolites is largely unexplored but potentially has profound developmental effects. Grafting experiments conducted in the early 1900s suggested that alkaloids move from the site of manufacture (the root) to the aerial tissue.¹⁴ More recent grafting experiments showed that root synthesised metabolites, perhaps carotenoids, regulate shoot development,^15,16 flowering inducers travel long distances,¹⁷ and phytohormones are translocated (reviewed in ref. ¹⁸).We recently showed that flavonoids moved long distances in Arabidopsis using several approaches.¹⁹ The roots of Arabidopsis seedlings grown in complete darkness do not accumulate flavonoids⁵ since expression of early genes encoding enzymes of flavonoid biosynthesis are light dependent.²⁰ Yet, flavonoids accumulate in root tips of seedlings with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain (diphenyl boric acid 2-amino ethyl ester [DPBA]), and localised aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating that early intermediates in the flavonoid pathway can move long distances. This was confirmed by time-course fluorescence experiments and HPLC. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application mid-root or to cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol and dihydroquercetin were taken up at the root tip, mid-root or through cotyledons and travelled long distances via cell-to-cell movement to distal tissues followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Uptake of flavonoids at the root tip was inhibited by glybenclamide, a specific inhibitor of ABC type transporters²¹ suggesting a possible role for transporters of this class in the movement of flavonoids.To show that endogenous flavonoids are capable of long distance movement, we performed reciprocal butt grafting between tt4 and wild-type seedlings.²² In these experiments we asked whether flavonoids moved from wild-type tissues to flavonoid-deficient tissues of tt4. DPBA fluorescence detection was used to detect flavonoid movement into tt4 tissues.¹⁹ Seedlings were grafted and grown on filter paper in Petri dishes for 8 d. The seedlings were then transferred to equal parts sand, perlite and vermiculite to avoid the possibility of uptake of pre-existing flavonoids that may be natural soil components. After 14 d, the seedlings were stained with DPBA. When tt4 roots were grafted to tt4 shoots, the samples showed dim greenish autofluorescence in roots and only red chlorophyll fluorescence in the shoot. When either tt4 roots or shoots were grafted onto wild-type shoots or roots, respectively, the tt4 tissues showed bright yellow DPBA fluorescence (Fig. 1). The results of these experiments clearly showed that endogenous flavonoids moved across the graft to the reciprocal tissue. The flavonoid movement is specific to certain tissues, as flavonoids are not transported into the seeds developing on tt4 shoots grafted on wild-type roots, which retain the transparent testa phenotype. Although flavonoids clearly travelled from wild-type root tissue to mutant shoots, they were not capable of complementing the seed colour defect of tt4. In addition, adding naringenin to tt4 plants either to the media, or to the soil, also did not complement the seed colour phenotype in tt4 (data not shown). Recent research by Hsieh and Huang²³ may account for this inability to complement seed colouration, as flavonoids in the Brassicaceae end up in the pollen coat rather than the testa. The testa tissue derives from ovular tissue,²⁴ and thus is maternal in origin.Open in a separate windowFigure 1Grafting shows flavonoid movement occurs across grafts. Reciprocal grafting between wild type and tt4 indicated flavonoid movement from the flavonoid producing tissue to the chalcone synthase-deficient tissue. The order of the graft is indicated by the left legend as aerial tissue over root tissue in the graft. Micrographs are DPBA stained tissue excited with 488 nm wavelength. The tt4/tt4 control graft shows no flavonoids are present, even though a wound has occurred on the leaf which generally exacerbates flavonoid fluorescence. Scale bar = 100 µm. Green fluorescence is from kaempferol and gold from quercetin. The red fluorescence is from chlorophyll.The complexity of the flavonoid biosynthetic pathway and the large number of modified flavonoids that can be made through the complex series of glycosylation reactions suggests that distinct flavonoid molecules may have unique function. To fully understand these molecules, it is necessary to dissect the synthesis pathways for these glycosylated flavonoids. Two unnamed flavonoid glycoside mutants isolated in 1998,²⁵ have profound developmental phenotypes, supporting this hypothesis. These mutations resulted in whorled cauline leaves on inflorescences and double the number of rosette leaves. Our lab is in the process of determining if other phenotypes exist in flavonoid mutants.A critical feature of the observations of flavonoid movement is understanding the biological context of this movement. First, a number of recent studies reported physiological functions of flavonoids in roots, ranging from modulation of auxin transport and root gravitropism,^5–8 to nodulation³ and root branching,⁸ while it is clear that flavonoid synthesis is absent in dark-grown seedlings.⁵ Yet, for flavonoids to function in roots of plants grown in soil, the light signal and/or flavonoid precursors must travel to the roots. Additionally, transient flavonoid accumulation has been reported in roots reoriented relative to the gravity vector.⁶ For flavonoids to transiently accumulate at the root tip (at 2 hours after reorientation) and to return to lower levels (within 2 additional hours), suggests that more than flavonoid synthesis is regulated. Perhaps this transient flavonoid accumulation requires localized enzyme activation and transport mechanisms. As flavonoid transport is inhibited by a compound that blocks ABC transporters, which include the newly identified auxin transporters of the MDR/PGP class, perhaps there are connections between flavonoid and auxin transport that allow this transient accumulation. A more detailed understanding of this role of flavonoid movement in controlling plant development awaits additional experimentation.     

Internal flavonoid patterns of diploids and tetraploids of two exudate chemotypes of Pityrogramma triangularis (Kaulf.) Maxon

The internal flavonoid patterns of Pityrogramma triangularis fronds were found to distinguish diploids and tetraploids of two exudate chemotypes (the ceroptin and the kaempferol methyl ether types). The flavonoid data suggest that the tetraploid-kaempferol methyl ether chemotype is of alloploid origin involving the two diploid chemotypes because the flavonoid pattern for the former represents a pattern additive of the two diploids, while the tetraploid-ceroptin chemotype may be of autoploid origin, as deduced from the similarity of the diploids and tetraploids.     

Crystal structure of Medicago truncatula UGT85H2--insights into the structural basis of a multifunctional (iso)flavonoid glycosyltransferase

(Iso)flavonoids are a diverse group of plant secondary metabolites with important effects on plant, animal and human health. They exist in various glycosidic forms. Glycosylation, which may determine their bioactivities and functions, is controlled by specific plant uridine diphosphate glycosyltransferases (UGTs). We describe a new multifunctional (iso)flavonoid glycosyltransferase, UGT85H2, from the model legume Medicago truncatula with activity towards a number of phenylpropanoid-derived natural products including the flavonol kaempferol, the isoflavone biochanin A, and the chalcone isoliquiritigenin. The crystal structure of UGT85H2 has been determined at 2.1 A resolution, and reveals distinct structural features that are different from those of other UGTs and related to the enzyme's functions and substrate specificities. Structural and comparative analyses revealed the putative binding sites for the donor and acceptor substrates that are located in a large cleft formed between the two domains of the enzyme, and indicated that Trp360 may undergo a conformational change after sugar donor binding to the enzyme. UGT85H2 has higher specificity for flavonol than for isoflavone. Further substrate docking combined with enzyme activity assay and kinetic analysis provided structural insights into this substrate specificity and preference.