Flavonoids and UV photoprotection in Arabidopsis mutants.

Wild-type Arabidopsis L. leaves exposed to low ultraviolet-B (UVB) conditions contained predominantly kaempferol glycosides, with low levels of quercetin glycosides. The flavonoid level doubled on treatment with UVB and an increase in the ratio of quercetin: kaempferol was observed. These results suggest that flavonols protect Arabidopsis plants from UVB damage, and indicate that the flavonoid 3'-hydroxylase (F3'H) enzyme, which converts dihydrokaempferol to dihydroquercetin, may play a crucial role. The tt7 mutant lacks this gene and, after treatment with sub-ambient UVB, contained kaempferol glycosides exclusively, to a level of total flavonols similar to that in wild-type Arabidopsis. Total flavonols after enhanced UVB treatment were higher in tt7 than in similarly treated wild-type plants, and only kaempferol glycosides were detected. Despite this high level, tt7 plants were less tolerant of UVB radiation than wild-type plants. These observations suggests that kaempferol is a less effective photoprotectant than quercetin. The chalcone isomerase (CHI) mutant (tt5) surprisingly did not accumulate naringenin chalcone, and this suggests that the mutation may not be restricted to the CHI gene alone. The concentration of hydroxycinnamic acid derivatives did not change with UVB treatment in most varieties indicating that their role in UV photoprotection may be subordinate to that of the flavonoids.

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.

Flavonoid characters contributing to the taxonomic revision of the Hebe parviflora complex.

Comparative flavonoid chemistry is a key element of a multidisciplinary study aimed at a revision of the genus Hebe, New Zealand's largest genus of flowering plants. One aspect of this study has been an investigation of the Hebe parviflora complex. A recent botanical paper on this topic marshalls generalised flavonoid data and morphological characters to support the recognition of two species in this complex, Hebe stenophylla and Hebe parviflora, which are clearly distinguishable from each other and from the related Hebe traversii and Hebe strictissima. A detailed study of the flavonoid chemistry and the distributional data used to support these conclusions are presented here. Six new compounds have been isolated in this study, including 6-hydroxyapigenin-7-O-beta-[2-O-beta-xyloxyloside] and-7-O-beta-[2-O-beta-xyloglucoside], 6-hydroxyluteolin-7-O-beta-[2-O-beta-xyloxyloside] and, luteolin-, 6-hydroxyluteolin- and 4'-O-methylluteolin-7-O-beta-[6-O-beta-xyloglucoside]. Other flavonoids include apigenin and luteolin 7- and 4'- mono-, di- and possibly tri-O-glycosides, 8-hydroxyluteolin 7- and 8-O-glucosides, and kaempferol and quercetin 3-O-mono- and di-glycosides. New structure assignments are supported with detailed 1H and 13C NMR data, including HMQC and HMBC measurements.

Anthocyanic vacuolar inclusions--their nature and significance in flower colouration.

The petals of a number of flowers are shown to contain similar intensely coloured intravacuolar bodies referred to herein as anthocyanic vacuolar inclusions (AVIs). The AVIs in a blue-grey carnation and in purple lisianthus have been studied in detail. AVIs occur predominantly in the adaxial epidermal cells and their presence is shown to have a major influence on flower colour by enhancing both intensity and blueness. The latter effect is especially dramatic in the carnation where the normally pink pelargonidin pigments produce a blue-grey colouration. In lisianthus, the presence of large AVIs produces marked colour intensification in the inner zone of the petal by concentrating anthocyanins above levels that would be possible in vacuolar solution. Electron microscopy studies on lisianthus epidermal tissue failed to detect a membrane boundary in AVI bodies. AVIs isolated from lisianthus cells are shown to have a protein matrix. Bound to this matrix are four cyanidin and delphinidin acylated 3,5-diglycosides (three, new to lisianthus), which are relatively minor anthocyanins in whole petal extracts where acylated delphinidin triglycosides predominate. Flavonol glycosides were not bound. A high level of anthocyanin structural specificity in this association is thus implied. The specificity and effectiveness of this anthocyanin "trapping" is confirmed by the presence in the surrounding vacuolar solution of only delphinidin triglycosides, accompanied by the full range of flavonol glycosides. "Trapped" anthocyanins are shown to differ from solution anthocyanins only in that they lack a terminal rhamnose on the 3-linked galactose. The results of this study define for the first time the substantial effect AVIs have on flower colour, and provide insights into their nature and their specificity as vacuolar anthocyanin traps.

Cell wall sited flavonoids in lisianthus flower petals.

Flavonoids are considered to be located predominantly in the vacuoles of epidermal cells and in the cuticular wax of terrestrial plants. However, recent reports have suggested that flavonoids may also reside elsewhere in the cells of green leaves. In the present study of lisianthus flower petals, it is demonstrated that ca. 30% of the whole petal flavonol glycosides are located in the cell wall. These flavonol glycosides are distinguished from the vacuolar glycosides in that they lack acylation. Evidence from light and confocal microscopy studies is corroborated by HPLC analyses of isolated protoplasts and cell wall digests, these having been produced by enzymic treatment of epidermal peels. This is the first report of the occurrence of flavonoids in petal cell walls, and it describes novel methodology for such studies.

Functional role of anthocyanins in the leaves of Quintinia serrata A. Cunn.

The protective functions that have been ascribed to anthocyanins in leaves can be performed as effectively by a number of other compounds. The possibility that anthocyanins accumulate most abundantly in leaves deficient in other phytoprotective pigments has been tested. Pigment concentrations and their histological distribution were surveyed for a sample of 1000 leaves from a forest population of Quintinia serrata, which displays natural polymorphism in leaf colour. Eight leaf phenotypes were recognized according to their patterns of red coloration. Anthocyanins were observed in almost all combinations of every leaf tissue, but were most commonly located in the vacuoles of photosynthetic cells. Red leaves contained two anthocyanins (Cy-3-glc and Cy-3-gal), epicuticular flavones, epidermal flavonols, hydroxycinnamic acids, chlorophylls, and carotenoids. Green leaves lacked anthocyanins, but had otherwise similar pigment profiles. Foliar anthocyanin levels varied significantly between branches and among trees, but were not correlated to concentrations of other pigments. Anthocyanins were most abundant in older leaves on trees under canopies with south-facing gaps. These data indicate that anthocyanins are associated with photosynthesis, but do not serve an auxiliary phytoprotective role. They may serve to protect shade-adapted chloroplasts from brief exposure to high intensity sunflecks.

Novel anthocyanins produced in petals of genetically transformed lisianthus.

The structures of the pigments in the deep purple flowers of a lisianthus line, transformed with a UDP-glucose:flavonoid-3-O-glucosyltransferase cDNA from Antirrhinum majus, have been studied using paper chromatography, HPLC and 1H and 13C NMR spectroscopy involving the use of 1H-1H COSY and TOCSY techniques. The transgenic line is shown to have produced the new anthocyanins delphinidin-3-O-beta-D-(6-O-alpha-L -rhamnopyranosylglucopyranoside)-5-O-beta-D-[6-E (and Z)-p-coumaroylglucopyranoside] and delphinidin -3-O-beta-D-glucopyranoside-5-O-beta-D-(6-O-E-p -coumaroylglucopyranoside) in addition to those found in the untransformed control plants.

Isoswertiajaponin 2''-O-beta-arabinopyranoside and other flavone-C-glycosides from the Antarctic grass Deschampsia antarctica.

The major falvonoid constituents of the Antarctic grass, Deschampsia antarctica, are shown to be the C-glycosylfavones, isoswertiajaponin (7-O-methylorientin) 2''-O-beta-arabinopyranoside and orientin. These are accompanied by lower levels of orientin 2''-O-arabinopyranoside, isoswertisin (7-O-methylvitexin) 2''-O-beta-arabinopyranoside, acylated derivatives, isoswertisin, isoswertiajaponin and tricin. A preliminary study suggests that the overall level of flavonoids in Deschampsia increases during the Antarctic mid-summer.

Lisianthus flavonoid pigments and factors influencing their expression in flower colour.

NMR, MS and analytical data are cited in support of the newly defined complete structures of the major flavonoid pigments and copigments in lisianthus flowers. The copigments newly characterized and found in flowers of all colours are kaempferol-3-O-beta-D-[6-O-rhamnopyranosyl-4-O-E-p- coumaroylgalactopyranoside]-7-O-alpha-L-rhamnopyranoside, its Z-isomer and by analogy, the lesser isorhamnetin and myricetin equivalents. Purple flower pigments with newly defined structures are: delphinidin-3-O-beta-D-[6-O-alpha- L-rhamnopyranosylgalactopyranoside] 5-O-beta-D-[6-E-p- coumaroylglucopyranoside], its Z-isomer, and by analogy the lesser cyanidin equivalent, together with delphinidin-3-O-beta-D-galactopyranoside-5-O-beta-D-[6-E-p- coumaroylglucopyranoside], its Z-isomer, and by analogy the lesser cyanidin equivalent. Different pigment/copigment compositions are shown to account for the basic colour differences between white, cream, pink, mauve and purple flowers, but other factors involved in stabilizing and fine-tuning the colours are pigment concentration, copigmentation and pH control.

Kaempferol-3-O-glucosyl(1-2)rhamnoside from Ginkgo biloba and a reappraisal of other gluco(1-2, 1-3 and 1-4)rhamnoside structures.

A kaempferol-3-O-glucorhamnoside from Ginkgo biloba is defined as the 3-O-alpha-L-[ beta-D-glucopyranosyl(1-2)rhamnopyranoside] on the basis of 2D NMR evidence. Complete assignments of the 1H and 13C NMR spectra of this compound and of its known p-coumaroyl derivative are presented for the first time. The NMR distinctions of 1-2, 1-3 and 1-4 linked glucopyranosylrhamnopyranosides are discussed and indicate (i) that the 13C NMR assignments for one published gluco(1-3)rhamnoside are in need of modification, (ii) that the published structure of hordenine-O-[6-O-t-cinnamoyl-beta-glucosyl(1-4)-alpha-rhamnoside] from Selaginella doederleinii is not distinguished from the 1-3 linked glucorhamnoside structure, and (iii) that the 8-prenylkaempferol-3-O-[glucosyl(1-4)rhamnoside]-7-O-glucoside and the equivalent 4'-O-methylated xylosyl(1-4)rhamnoside from Epimedium pubescens and E. washanense, respectively, are (1-2)-linked.