Stephen J. Bloor

An increase in the luteolin : Apigenin ratio in Marchantia polymorpha on UV-B enhancement

Abstract The effects of varying the UV-B component in ambient light on the liverwort, Marchantia polymorpha are reported. Plants grown under conditions of ambient light, ambient light lacking UV-B, and ambient light with a 25% enhancement of incident UV-B showed, with increasing levels of UV-B, a decrease in growth rate, a decrease in the production of gemmae cups and an increase in the proportion of dead thallus. Thallus surviving after three months of summer growth under these conditions showed no statistically significant increase in flavonoid levels with increasing UV-B levels. However, HPLC monitoring of individual flavonoids (all of which are apigenin and luteolin glucuronides) revealed a strong correlation between increasing UV-B levels and an increase in the ratio of luteolin to apigenin glycosides. It is considered unlikely that this change has significantly altered the UV-B screening effectiveness of the flavonoids. Rather, an improved level of antioxidant defence, or a more effective dissipation of absorbed UV energy, are proposed as possible UV-B protectant benefits to the plant.

UVB RADIATION INDUCED INCREASE IN QUERCETIN : KAEMPFEROL RATIO IN WILD-TYPE AND TRANSGENIC LINES OF PETUNIA

The use of genetically modified plants offers unique opportunities to study the role of specific flavonoids in plant UVB protection. Along with a parental wild‐type Mitchell Petunia, two transgenic lines with altered flavonoids were also examined; Lc with enhanced levels of antho‐cyanins due to the action of a maize flavonoid regulatory gene Leaf color, and AFLS that carries an antisense fla‐vonol synthase construct and is known to have reduced flavonol levels in flowers. All three lines were grown in near ambient sunlight, sunlight lacking UVB (280–320 nm) radiation and sunlight with 25% added UVB. Ultra‐violet‐B radiation induced significant reductions in the rates of leaf expansion and seedling growth in all three lines. The presence of anthocyanins did not appear to afford Lc plants any special protection from UVB. Ul‐traviolet‐B treatment induced increases in total flavonol content in young plants of all three lines, and this effect decreased with increasing leaf age. Notably, increasing UVB levels led to an increase in the ratio of quercetin: kaempferol with all three cultivars. The AFLS transgenic, contrary to expectations based on its genetic construction, had normal levels of flavonols in the leaves and the highest Q:K ratio of the three cultivars. This transgenic was the least susceptible to UVB, which may indicate an enhanced protective role for quercetin. Because both quercetin and kaempferol have similar UVB screening properties, quercetin may exert this role by other means.

Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase

Flavonoids, in particular the anthocyanins,are responsible for flower colour in manyspecies. The dihydroflavonols represent abranch point in flavonoid biosynthesis,being the intermediates for production ofboth the coloured anthocyanins, through theaction of the enzyme dihydroflavonol4-reductase (DFR), and the colourlessflavonols, produced by flavonol synthase(FLS). In this study the white-flowered,flavonol accumulating Mitchell line ofpetunia was used as a model to examine theinteraction between DFR and FLS enzymeactivities and possibilities forredirecting flavonoid biosynthesis awayfrom production of flavonols and towardsanthocyanins. Introduction of a 35SCaMV-DFR sense transgene construct causedthe production of anthocyanins, resultingin a pink-flowered phenotype. Furthermore,inhibition of FLS production throughintroduction of an FLS antisense RNAconstruct also led to anthocyaninproduction and a pink-flowered phenotype. A combination of both transgenes gave thehighest level of anthocyanin formation. Anthocyanins were produced in the DFR-senseand FLS-antisense transgenic lines in spiteof the greatly reduced levels of geneexpression in the Mitchell line for threeenzymes late in anthocyanin biosynthesis,anthocyanindin synthase, UDP-glucose:flavonoid 3-O-glucosyltransferase andUDP-rhamnose: anthocyanidin-3-glucosiderhamnosyltransferase. Thus, the level ofgene activity required for visibleanthocyanin formation is much lower thanthe high levels normally induced duringpetal development. Altering the balancebetween the DFR and FLS enzyme activities,using genetic modification, may be a usefulstrategy for introducing or increasinganthocyanin production in target ornamentalspecies.

The structure of the major anthocyanin in Arabidopsis thaliana

The major anthocyanin in the leaves and stems of Arabidopsis thaliana has been isolated and shown to be cyanidin 3-O-[2-O(2-O-(sinapoyl)-beta-D-xylopyranosyl)-6-O-(4-O-(beta-D-glucopyranosyl)-p-coumaroyl-beta-D-glucopyranoside] 5-O-[6-O-(malonyl) beta-D-glucopyranoside]. This anthocyanin is a glucosylated version of one of the anthocyanins found in the flowers of the closely related Matthiola incana.

An antisense chalcone synthase cDNA leads to novel colour patterns in lisianthus (Eustoma grandiflorum) flowers

Three cultivars of lisianthus (Eustoma grandiflorum (Grise.)) were transformed with a homologous antisense CHS cDNA via Agrobacterium-mediated transformation. Over 50% of the transgenics derived from the purple flowering lines exhibited an altered flower colour pattern ranging from small streaks of white on the wild-type purple background through to completely white flowers. A significant portion of the transgenic lines showed unstable phenotypes. Northern and biochemical analysis showed that the altered flower patterns were associated with a loss of CHS gene transcript and a corresponding loss of CHS enzyme activity. In the white flowering line the level of total flavonoids was reduced to ca. 2.0% of the wild-type level. Some of the transgenic plants also exhibited alterations in flower form such as the formation of frilled petal tips and reduced flower opening. Several of the new patterned lines are being evaluated for stability and possible commercial release.

Variation in the ability of the maize Lc regulatory gene to upregulate flavonoid biosynthesis in heterologous systems

Abstract We have previously shown that overexpression of the maize basic helix-loop-helix (bHLH) regulatory gene, Leaf colour (Lc) in petunia enhanced pigmentation through the upregulation of the flavonoid biosynthetic pathway (Bradley et al. Plant J. (13) (1998) 381–392) [1] . Here we report on the effect of Lc expression in two popular ornamental species, lisianthus (Eustoma grandiflorum) and a regal pelargonium cultivar (Pelargonium X domesticum Dubonnet). A number of transformants expressing the Lc cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter were generated in three different lisianthus cultivars, including purple, pink and cream flowered lines, and the pelargonium cultivar Dubonnet. Unlike transgenic Lc petunia, which had enhanced pigmentation in foliage and flowers, no visible phenotypic alteration in vegetative or floral pigmentation was observed in either the pelargonium or lisianthus lines expressing Lc. A detailed analysis of flavonoid content and expression of a number of flavonoid biosynthetic genes in the leaves of these lines indicated that, unlike in petunia, Lc alone was unable to transcriptionally upregulate the flavonoid biosynthetic genes of lisianthus or pelargonium. This suggests there may be a divergence of the regulatory mechanisms in different dicots and that a combination of introduced bHLH and MYB factors may be required to increase pigmentation in some plant species. This report extends the study of bHLH regulatory gene action in several Solanaceae and a single Brassiceae species to include members of the Gentianaceae and Geraniaceae.

Blue flower colour derived from flavonol—anthocyanin co-pigmentation in Ceanothus papillosus

The pigments responsible for the blue flower colour of Ceanothus papillosus have been identified. The colour arises from co-pigmentation between the anthocyanins and flavonols. The major anthocyanins have been identified from NMR spectroscopy and degradation studies as the novel acylated delphinidin glycosides, delphinidin 3-O-rutinoside 7-O-(6-O-p-coumaroylglucoside 3′-O-glucoside and delphinidin 3-O- rutinoside 7,3′-di-O-(6-O-p-coumaroylglucoside), and the major flavonol is kaempferol 3-O-xylosyl-(1 → 2)- rhamnoside. The co-pigmentation effect appears to be quite specific, and does not occur to the same extent with other, more common, flavonols. The effect is particularly notable in that an extraordinary, long wavelength visible absorbtion maxima at 680 nm is produced which confers additional blueness. This is proposed to arise from a supramolecular complex of high stoichiometry.

A macrocyclic anthocyanin from red\mauve carnation flowers

Abstract Flowers of the red\mauve carnation cultivars ‘‘Kortina Chanel’’ and ‘‘Purple Torres’’ contain a unique macrocyclic anthocyanin pigment, a malylated cyanidin 3,5-diglucoside in which the malyl group is linked to both sugars. The native anthocyanin readily undergoes ring opening to yield cyanidin 3- O -(6- O -malyl glucoside)-5- O -glucoside. This lability was found to be due to the inherent instability of the malyl interglycosidic bridge. In this paper we report the structure elucidation of the native anthocyanin, and the ring opened product.

Identification of flavonol and anthocyanin metabolites in leaves of petunia Mitchell and its LC transgenic

Abstract The leaves of Petunia Mitchell, a model species employed for transgenic manipulation, contain a wider array of acylated flavonol glycosides than occur in the petals. Three new acylated flavonol glycosides are described, kaempferol-3- O -(2- O -feruloyl- β - d -glucosyl(1→2)6- O -malonylglucoside), quercetin-3- O -(2- O -caffeoyl- β - d -glucosyl(1→2)6- O -malonylglucoside), and quercetin-3- O -(2- O -feruloyl- β - d -glucosyl(1→2)glucoside). A transgenic Mitchell line expressing the maize Leaf colour (Lc) cDNA had enhanced levels of anthocyanins, particularly in their leaves. These anthocyanins were determined to be the same acylated petunidin glycosides as those which produce a slight red colouration in the tube of the flowers of Petunia Mitchell.

Flavonoid and carotenoid pigments in flower tissue of Sandersonia aurantiaca (Hook.)

Luteolin, cryptoxanthin and zeaxanthin were detected as the major pigments present in mature flowers of Sandersonia aurantiaca (Hook.). Both the flavonoids and carotenoid pigments were examined at four stages of flower development from green flower bud to senescent flower prior to tepal necrosis. The major component of the orange colour of the Sandersonia flowers is due to the presence of the carotenoids zeaxanthin and cryptoxanthin, both predominantly present in the esterified form. The main change during development is the loss of chlorophyll, β-carotene and lutein, and the increase in zeaxanthin and cryptoxanthin as the flower matures from a green bud to an open flower. The major flavonoids present were luteolin and luteolin 7-O-glucoside. Flavonoid concentration decreased over the course of development to a mature flower but there was an increase in content per flower. The flavonoids were present in greater quantities than the carotenoids. However, as they absorb mostly in the UV region, they are not believed to contribute significantly to the observed colour.