Vacuolar Flavonoids Research Articles

Cloning and Expression of a Nonribosomal Peptide Synthetase to Generate Blue Rose.

Rose has been entwined with human culture and history. "Blue rose" in English signifies unattainable hope or an impossible mission as it does not exist naturally and is not breedable regardless of centuries of effort by gardeners. With the knowledge of genes and enzymes involved in flower pigmentation and modern genetic technologies, synthetic biologists have undertaken the challenge of producing blue rose by engineering the complicated vacuolar flavonoid pigmentation pathway and resulted in a mauve-colored rose. A completely different strategy presented in this study employs a dual expression plasmid containing bacterial idgS and sfp genes. The holo-IdgS, activated by Sfp from its apo-form, is a functional nonribosomal peptide synthetase that converts l-glutamine into the blue pigment indigoidine. Expression of these genes upon petal injection with agro-infiltration solution generates blue-hued rose flowers. We envision that implementing this proof-of-concept with obligatory modifications may have tremendous impact in floriculture to achieve a historic milestone in rose breeding.

Further Investigation into the Flavonoid Profile of Balsamorhiza macrophylla (Nutt.) A. Gray (Asteraceae)

<p class="jbls"><span lang="EN-GB">This research has identified the vacuolar flavonoids of <em>Balsamorhiza</em> <em>macrophylla</em> Nutt., which consist of Quercetin-3-O-galactoside, Quercetin-3-O-rutinoside, and Quercetin-7-O-glucoside. Exudate lipophilic flavonoids were also investigated. Results vary slightly from those of earlier study. This study shows the presence of Quercetin 3 - O - methyl ether, Quercetin 3,6,4'- O - trimethyl ether, Quercetin 3,4'-O-dimethyl ether, 3,3'-O-dimethyl ether and an unknown compound which absorbs at 290nm, possibly a flavanone. The first three compounds agree with earlier results, which also include Tamarexetin, Axillarin and Spinacetin. Workers had reported earlier that flavonoid profiles often vary due to environmental or ecological differences, and whether this applies to <em>Balsamorhiza</em> <em>macrophylla</em> is part of this investigation.</span></p>

Functional roles of flavonoids in photoprotection: New evidence, lessons from the past

We discuss on the relative significance of different functional roles potentially served by flavonoids in photoprotection, with special emphasis to their ability to scavenge reactive oxygen species (ROS) and control the development of individual organs and whole plant. We propose a model in which chloroplast-located flavonoids scavenge H2O2 and singlet oxygen generated under excess light-stress, thus avoiding programmed cell death. We also draw a picture in which vacuolar flavonoids in conjunction with peroxidases and ascorbic acid constitute a secondary antioxidant system aimed at detoxifying H2O2, which may diffuse out of the chloroplast at considerable rates and enter the vacuole following excess light stress-induced depletion of ascorbate peroxidase. We hypothesize for flavonols key roles as developmental regulators in early and current-day land-plants, based on their ability to modulate auxin movement and auxin catabolism. We show that antioxidant flavonoids display the greatest capacity to regulate key steps of cell growth and differentiation in eukaryotes. These regulatory functions of flavonoids, which are shared by plants and animals, are fully accomplished in the nM concentration range, as likely occurred in early land plants. We therefore conclude that functions of flavonoids as antioxidants and/or developmental regulators flavonoids are of great value in photoprotection. We also suggest that UV-B screening was just one of the multiple functions served by flavonoids when early land-plants faced an abrupt increase in sunlight irradiance.

Flavonoid Biosynthesis in Barley Primary Leaves Requires the Presence of the Vacuole and Controls the Activity of Vacuolar Flavonoid Transport

Barley (Hordeum vulgare) primary leaves synthesize saponarin, a 2-fold glucosylated flavone (apigenin 6-C-glucosyl-7-O-glucoside), which is efficiently accumulated in vacuoles via a transport mechanism driven by the proton gradient. Vacuoles isolated from mesophyll protoplasts of the plant line anthocyanin-less310 (ant310), which contains a mutation in the chalcone isomerase (CHI) gene that largely inhibits flavonoid biosynthesis, exhibit strongly reduced transport activity for saponarin and its precursor isovitexin (apigenin 6-C-glucoside). Incubation of ant310 primary leaf segments or isolated mesophyll protoplasts with naringenin, the product of the CHI reaction, restores saponarin biosynthesis almost completely, up to levels of the wild-type Ca33787. During reconstitution, saponarin accumulates to more than 90% in the vacuole. The capacity to synthesize saponarin from naringenin is strongly reduced in ant310 miniprotoplasts containing no central vacuole. Leaf segments and protoplasts from ant310 treated with naringenin showed strong reactivation of saponarin or isovitexin uptake by vacuoles, while the activity of the UDP-glucose:isovitexin 7-O-glucosyltransferase was not changed by this treatment. Our results demonstrate that efficient vacuolar flavonoid transport is linked to intact flavonoid biosynthesis in barley. Intact flavonoid biosynthesis exerts control over the activity of the vacuolar flavonoid/H(+)-antiporter. Thus, the barley ant310 mutant represents a novel model system to study the interplay between flavonoid biosynthesis and the vacuolar storage mechanism.

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.

Variations in lipophilic and vacuolar flavonoids among European Pulicaria species

Four European Pulicaria species, P. odora, P. paludosa, P. sicula and P. vulgare, were analysed for their surface and vacuolar constituents for comparison with previous data obtained for P. dysenterica. Each species had a distinct flavonoid pattern with notable differences between leaf and inflorescence. 6-Hydroxyflavonols were the major lipophilic components in all of the species and tissues except in the leaves of P. paludosa and P. vulgare, where scutellarein 6-methyl ether was the main constituent. In the leaves of P. sicula a more unusual flavone, 6-hydroxyluteolin 5,6,7,3′,4′-pentamethyl ether, was a major component. Pulicaria odora was distinguished by the presence of a series of methylated 6-hydroxykaempferol derivatives including a 3,5,6,7,4′-pentamethyl ether. Quercetagetin hexamethyl ether occurred in both tissues of P. sicula together with the 3,7,3,4′-tetra methyl ether and other quercetagetin derivatives, which were 5-methylated. Quercetagetin 3,7,3′-methyl ether was present in all species except P. odora. Flavonol glucuronides were characteristic vacuolar constituents of all the taxa studied. Two rare glycosides, patuletin and 6-hydroxykaempferol 6-methyl ether 7-glucuronides were identified in the inflorescence of P. odora. Pulicaria vulgaris, a rare plant of southern England, had the vacuolar flavonoid profile most similar to the other more abundant British plant, P. dysenterica.

Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration

It is widely accepted that the mix of flavonoids in the cell vacuole is the source of flavonoid based petal colour, and that analysis of the petal extract reveals the nature and relative levels of vacuolar flavonoid pigments. However, it has recently been established with lisianthus flowers that some petal flavonoids can be excluded from the vacuolar mix through deposition in the cell wall or through complexation with proteins inside the vacuole, and that these flavonoids are not readily extractable. The present work demonstrates that flavonoids can also be compartmented within the cell cytoplasm. Using adaxial epidermal peels from the petals of lisianthus ( Eustoma grandiflorum), Lathyrus chrysanthus and Dianthus caryophyllus, light and laser scanning confocal microscopy studies revealed a significant concentration of petal flavonoids in the cell cytoplasm of some tissues. With lisianthus, flavonoid analyses of isolated protoplasts and vacuoles were used to establish that ca 14% of petal flavonoids are located in the cytoplasm (cf. 30% in the cell wall and 56% in the vacuole). The cytoplasmic flavonoids are predominantly acylated glycosides (cf. non-acylated in the cell wall). Flavonoid aggregation on a cytoplasmic protein substrate provides a rational mechanism to account for how colourless flavonoid glycosides can produce yellow colouration in petals, and perhaps also in other plant parts. High vacuolar concentrations of such flavonoids are shown to be insufficient.

The role of lipophilic and polar flavonoids in the classification of temperate members of the Anthemideae.

Lipophilic and vacuolar flavonoids were separately identified in representative temperate species of the genera Anthemis, Chrysanthemum, Cotula, Ismelia, Leucanthemum and Tripleurospermum. The four Anthemis species investigated variously produced four main surface constituents, in leaf and flower: santin, quercetagetin 3,6,3'-trimethyl ether, scutellarein 6,4'-dimethyl ether and 6-hydroxyluteolin 6,3'-dimethyl ether. By contrast, surface extracts of disc and ray florets of the species of Chrysanthemum, Cotula, Ismelia, Leucanthemum and Tripleurospermum surveyed yielded five common flavones in the free state: apigenin, luteolin, acacetin, apigenin 7-methyl ether and chrysoeriol. Polar flavonoids were isolated and identified in leaf, ray floret and disc floret of all the above plants. Anthemis species were distinctive in having flavonol glycosides in the leaves, whereas the leaf flavonoids of the other taxa were generally flavone O-glycosides. The 3-glucoside and 3-rutinoside of patuletin were characterised for the first time from Anthemis tinctoria ssp. subtinctoria. Two new flavonol glycosides, the 5-glucuronides of quercetin and kaempferol, were obtained from the leaf of Leucanthemum vulgare, where they co-occur with the related 5-glucosides and with several flavone glycosides. The ray florets of these Anthemideae generally contain apigenin and/or luteolin 7-glucoside and 7-glucuronide, whereas disc florets have additional flavonol glycosides, notably the 7-glucosides of quercetin and patuletin and the 7-glucuronide of quercetin. A comparison of the flavonoid pattern encountered here with those previously recorded for Tanacetum indicate some chemical affinity between Anthemis and Tanacetum. Flavonoid patterns of the other five genera are more distinct from those of Tanacetum and suggest that those genera form a related group. All 14 species surveyed for their flavonoid profiles have distinctive constituents and the chemical data are in harmony with modern taxonomic treatments of the "Chrysanthemum complex" as a series of separate genera.

Geographical variation in the surface flavonoids of Pulicaria dysenterica.

Four chemical races were detected in Pulicaria dysenterica, when sampled within Europe, on the basis of the surface flavonoids present. One race uniquely contained quercetagetin 3,7-dimethyl ether and another 6-hydroxykaempferol 3,4'-dimethyl ether. A third race was based on plants having 6-hydroxykaempferol 3,7-dimethyl ether together with quercetagetin 3,7,3'-trimethyl ether. The fourth race contained the above two compounds, as well as quercetagetin 3,7,3',4'-tetramethyl ether and 6-hydroxykaempferol 3,7,4'-trimethyl ether. These lipophilic constituents were variously present on the surfaces of leaf, ray floret, disc floret and fruit. By contrast, the vacuolar flavonoid of all tissues and all races was uniformly quercetin 3-glucuronide. The kaempferol 3-glucoside previously reported in flowers was not detected. Of the lipophilic flavonoids newly reported from this plant, one 6-hydroxykaempferol 3,7,4'-trimethyl ether is new to nature.

Variations in lipophilic and polar flavonoids in the genus Tanacetum

Seven species of Tanacetum of different geographical origins were analysed in leaf and flower for their lipophilic and polar flavonoids and the results were compared with the patterns previously recorded in T. parthenium and T. vulgare. The lipophilic constituents are based generally on 6-hydroxykaempferol 3,6,4′-trimethyl ether and quercetagetin 3,6,3′-trimethyl ether, but up to 11 other surface flavonoids are present. Methyl ethers of scutellarein and 6-hydroxyluteolin are present in species with corymbose capitula. A rare carbomethoxyflavone reported earlier in T. microphyllum could not be detected. The dominant pattern of vacuolar flavonoids is based on apigenin and luteolin 7-glucuronides. 6-Hydroxyluteolin 7-glucoside, present in T. vulgare, also occurs in T. pseudoachillea. Chrysoeriol 7-glucuronide is present in T. parthenium, T. macrophyllum and T. corymbosum. By contrast, quercetin 7-glucuronide characterises T. parthenium, T. corymbosum and T. cinerariifolium. On the basis of combined data of lipophilic and polar constituents, it is clear that several structures are useful chemotaxonomic characters at the species level in a taxonomically complex genus.

The flavonoids of Tanacetum parthenium and T. vulgare and their anti-inflammatory properties.

The lipophilic flavonoids in leaf and flower of Tanacetum parthenium and T. vulgaris have been compared. While those of T. parthenium are methyl ethers of the flavonols 6-hydroxykaempferol and quercetagetin, the surface flavonoids of T. vulgare are methyl ethers of the flavones scutellarein and 6-hydroxyluteolin. Apigenin and two flavone glucuronides are surprisingly present in glandular trichomes on the lower epidermis of the ray florets of T. parthenium. The opportunity has been taken to revise the structures of the four 6-hydroxyflavonol methyl ethers of T. parthenium based on NMR measurements. These are now shown to be uniformly 6- rather than 7-O-methylated. Tanetin, previously thought to be a new structure, is now formulated as the known 6-hydroxykaempferol 3,6,4'-trimethyl ether. The vacuolar flavonoids of both plants are dominated by the presence of apigenin and luteolin 7-glucuronides; nine other glycosides were present, including the uncommon 6-hydroxyluteolin 7-glucoside in T. vulgare. When the major flavonol and flavone methyl ethers of the two plants were tested pharmacologically, they variously inhibited the major pathways of arachidonate metabolism in leukocytes. There were significant differences in potency, with the tansy 6-hydroxyflavones less active than the feverfew 6-hydroxyflavonols as inhibitors of cyclo-oxygenase and 5-lipoxygenase.

An 8-hydroxylated external flavone and its 8- O-glucoside from Becium grandiflorum

The major vacuolar flavonoid of a greenhouse-grown plant of Becium grandiflorum has been identified as the 8- O-glucoside of isothymusin (5,8,4′-trihydroxy-6,7-dimethoxyflavone), while the major external flavonoids in the diethyl ether surface wash of the same plant are found to be isothymusin and cirsimaritin (5,4′-dihydroxy-7,8-dimethoxyflavone). Although isothymusin is already known as a conversion product of thymusin (5,6,4′-trihydroxy-6,7-dimethoxyflavone) formed by means of the Wessely-Moser rearrangement, this is the first report of isothymusin as a plant constituent, both as aglycone and glycoside. The biogenetic and chemotaxonomic implications of the occurrence of these flavonoids in B. grandiflorum are briefly discussed.

Vacuolar flavonoids of rocky mountain maple, Acer glabrum torrey (Aceraceae)

Vacuolar flavonoids were investigated in Acer glabrum and compared to published data. The profile consists of two unidentified flavanones, two kaempferol 3- O-monoglycosides, two quercetin 3- O-monoglycosides, rutin and isorhamnetin. The presence of flavanones sheds new light on the relationship between A. glabrum and its supposed closest relatives. No differences were found in the profiles of infraspecific taxa.

Flavonoids of Umbellularia californica

The vacuolar flavonoid profile of Umbellularia californica consists of 3- O-mono-, di- and triglycosides of kaempferol, quercetin and isorhamnetin together with eriodictyol 7- O-glucoside. The leaf exudate contained no flavonoids but did exhibit a small amount of scopoletin. The two growth forms of this species, a shrubby form from dry habitats and a coastal forest tree, exhibited identical flavonoid profiles. These observations were taken to indicate that the flavonoid profile of this species became established before the two growth forms differentiated. The flavonol profile of Umbellularia is consistent with profiles reported for other members of the Lauraceae.

Vacuolar and excudate flavonoids of New Zealand Cassinia (Asteraceae: gnaphalieae)

The exudate and vacuolar flavonoid profiles of four species (five taxa) of New Zealand Cassinia were established. The exudate chemistry consists of chalcones, dihydrochalcones and flavanones, several of which lack B-ring oxygenation, and quercetin 3- O- and 7- O-methyl ethers. Vacuolar flavonoids include galangin, eriodictyol-7- O-methyl ether, and a series of kaempferol and quercetin glycosides. Several combinations of these compounds occur in the taxa studied. Limited common garden studies suggest that the flavonoid profiles from the different areas may be fixed. Differences in distribution of flavonoids could be taken as evidence for recognition of several taxa in contrast to the view that only one variable species exists in New Zealand.

Differences in flavonoid patterns between genera within the velloziaceae

Detailed flavonoid analysis of seven representative species of Aylthonia, Barbacenia, Burlemarxia and African and Madagascan Xerophyta have confirmed the paucity of surface flavonoids in these genera compared with Vellozia, where complex mixtures of lipophilic flavonoids are common. However, some simple flavonol O-methyl ethers including 6-methoxykaempferol 3- O-methyl ether were found in small amounts on the leaf surface of two Barbacenia species. In the subfamily Barbacenioideae, Aylthonia was distinguished from both Barbacenia and Burlemarxia in its vacuolar flavonoids by the presence of flavone mono- C-glycosides rather than flavone di- C-glycosides and Pleurostima is unique in lacking glycoflavones but possessing 6-hydroxyflavones. Complex mixtures of flavonol mono- O- and di- O-glycosides were found within the leaves of Barbacenia and Burlemarxia species, while only quercetin monoglycosides were detected in Aylthonia riedeliana. In Barbacenia conicostigma, the 3-apiosylgalactosides and 3-apiosylglucosides of quercetin and isorhamnetin were characterized; the former two compounds were present also in Burlemarxia spiralis. In Xerophyta eglandulosa var. eglandulosa from Madagascar an isomeric quercetin 3-apiosylglucoside occurred. The Madagascan Xerophyta species were distinguished from the African Xerophyta species by the absence of glycoflavones and presence of isorhamnetin glycosides, while in X. dasylirioides quercetin 3-methyl ether and quercetin di- and trimethyl ethers were identified free within the leaf. Similarly, in Barbacenia conicostigma four flavonol methyl ethers, as well as kaempferol and chrysoeriol occurred in the free state. However, X. retinervis from South Africa was exceptional in producing 6- C-methyl quercetin 3-methyl ether, chrysoeriol 7-glucoside and both flavone mono- and di- C-glycosides within the leaf. The present results confirm that flavonoid patterns are useful in determining generic limits within the family.

Variations in the lipophilic and vacuolar flavonoids of the genus Vellozia

Five lipophilic flavonoids were identified in Vellozia aff. scoparia: kaempferol, kaempferol 3-methyl ether, luteolin, chrysoeriol and a new compound, 6-prenylherbacetin 3,8-dimethyl ether. Five such substances were identified in V. phalocarpa: luteolin, chrysoeriol, eriodictyol, 6- C-methylquercetin 3-methyl ether and 8- C-methylquercetagetin 3,6,7,4′-tetramethyl ether. Five compounds were identified in V. laevis: velloquercetin 3,3′-dimethyl ether, 8- C-methyl-6-hydroxykaempferol 3,6,7-trimethyl ether and the 3,6,7,3′- and 3,6,7,4′-tetramethyl ethers of 8- C-methylquercetagetin. A method for distinguishing these latter two isomers is described. All three species also contained a range of vacuolar flavone O- and C-glycosides; two contained a new conjugate of orientin, the 7-caffeate. These lipophilic and vacuolar flavonoid patterns were compared with those of seven other Vellozia species and the results used to prepare similarity indices within the genus. Although all 10 species have distinctive profiles, there is no evidence to suggest that any of these species should be transferred to a different genus.

Ten isoprenylated and C-methylated flavonoids from the leaves of three Vellozia species

Eight new and two known lipophilic flavonoids were isolated from the leaf surface of Vellozia coronata, V. glabra and V. nanuzae. Four are flavanones: velloeriodictyol [2″-isopropenyldihydrofurano(4″,5″:6,7)-eriodictyol], 6,8-diprenyleriodictyol, 6-(3-hydroxyisopentanyl) eriodictyol and 6- C-methyleriodictyol 7,3′,4′-trimethyl ether. Two are flavonols, the 6-prenyl derivatives of quercetin 3-monomethyl ether and of quercetin 3,7-dimethyl ether. Two new C-methyl ethers, namely 8- C-methylquercetagetin 3,6,3′-trimethyl ether and 8- C-methyl-6-hydroxykaempferol 3,6,7-trimethyl ether, were accompanied by the known 3,6,7-trimethyl ether and 3,6,7,3′-tetramethyl ether of 8- C-methylquercetagetin. Also present in the lipophilic fraction is a resveratrol dimethyl ether mixture, which has considerable antifungal activity. The vacuolar flavonoids of the same three Vellozia species were variously identified as tricin 5-glucosisde, five orientin-based C-glycosides, the 7-glucoside and 7-rhamnosylglucoside of 6-hydroxyluteolin, the 7-glucoside of pleurostimin and the 6-glucoside of its 7-methyl ether. The chemosystematic significance of these findings is briefly discussed.

Six further lipophilic flavonols from the leaf of Vellozia stipitata

Six new lipophilic flavonols have been characterized from the leaf of Vellozia stipitata: vellokaempferol 3,5- dimethyl ether, velloquercetin 3-methyl ether and 3,5,3′-trimethyl ether, 8- C-methylvellokaempferol 3,5-dimethyl ether, 8- C-methylvelloquercetin 3-methyl ether and 3,5,3′-trimethyl ether. A seventh compound present is identical to 5,4′-dihydroxy-3,6,7,3′-tetramethoxy-8- C-methylflavone, recently detected in V. lilacina and V. aff. epidendroides. Vacuolar flavonoids in the leaf include iso-orientin 7-glucoside, iso-orientin, an orientin O-glucoside and a vicenin derivative. This pattern of flavonoid accumulation is compared with that present in the leaf of V. streptophylla.

Flavonoids and Affinities of Greyiaceae with a Discussion of the Occurrence of B-Ring Deoxyflavonoids in Dicotyledonous Families

The leaf surface flavonoid profiles of all three species of Greyia, the sole genus in Greyiaceae, have been shown to consist of a mixture of flavones, flavanones, dihydroflavonols and flavonols, several of which lack B-ring substitution. Several of these compounds were reported as components of Escallonia, a genus thought by some to be related to Greyiaceae. The vacuolar flavonoid profile consists of kaempferol and quercetin 3-0-mono- and diglycosides. Whereas the vacuolar flavonoids are not significant taxonomically, the identification of B-ring deoxyflavonoids as major components of leaf surface exudates of Greyia adds to the list of characters by which Greyiaceae and Melianthaceae can be distinguished. The widespread occurrence of B-ring deoxyflavonoids in dicot families reduces their usefulness for assessing relationships at higher taxonomic levels. The South African endemic, monogeneric family Greyiaceae Hutchinson consists of three species, Greyia flanaganii H. Bolus, G. sutherlandii Hook. & Harv. and G. radlkoferi Szyszyl. Greyia sutherlandii, the Natal bottlebrush, and G. radlko- feri, the mountain bottlebrush, are small, shrub- by, deciduous trees that occur principally in the Drakenberg Mountains. They are very similar and tend to intergrade. Greyia flanaganii, limited in occurrence to the eastern Cape Province, dif- fers from the others in floral features and is evergreen (Steyn 1974; Dahlgren and van Wyk 1988).