Environmental and developmental effects on the biosynthesis of UV-B screening pigments in Scots pine (Pinus sylvestris L.) needles

The major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles are flavonol 3-o-glycosides (F3Gs) esterified with hydroxycinnamic acids at positions 3" and 6". Acylation is the last step in biosynthesis and is catalysed by position-specific hydroxycinnamoyl transferases (3" and 6"HCT). The UV-B dependence of these enzyme activities was studied in primary needles of Scots pine seedlings grown under different UV-B conditions in environmentally controlled sun simulators. 6"HCT activity was induced upon UV-B irradiation while 3"HCT activity was not induced but showed high constitutive values. To investigate the biosynthesis of diacylated F3Gs during needle development under natural conditions, the HCT activities and metabolite contents were analysed in needles of field-grown mature pine trees. Accumulation of diacylated compounds as well as of 6"HCT activity occurred transiently in the first year of needle development only. In contrast, 3"HCT activity exhibited broad maxima in two consecutive years during needle growth. The data suggest that acylated F3Gs are first formed as soluble compounds which are then translocated into the cell wall to be bound by their hydroxycinnamoyl residues.     

Flavonol glycosides of Phlomis spectabilis

Four flavonol glucosides, one new, have been isolated from a methanolic extract of Phlomis spectabilis. Their structures were established as the 3-glucosides and 3-(6″-(E)-p-coumaroyl)glucosides of kaempferol and of kaempferol 7,4′-dimethyl ether.     

Four acylated flavonol glycosides from leaves of Strychnos variabilis

Four new acylated flavonol glycosides have been isolated and identified from the leaves of Strychnos variabilis: quercetin 3-(4″-trans-p-coumaroyl)robinobioside-7-glucoside (variabiloside A) and its cis derivative (variabiloside B), kaempferol 3-(4″-trans-p-coumaroyl)robinobioside-7-glucoside (variabiloside C) and its cis derivative (variabiloside D).     

Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.)

Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at positions 3' and 6' are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles. The last steps in the biosynthesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O-glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specificities for positions 3', 4' or 6'. The positions of the acyl groups were identified by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelectric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3'- and 6'HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6' followed by acylation at position 3'. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280-315 nm) radiation.     

Structurally different flavonol glycosides and hydroxycinnamic acid derivatives respond differently to moderate UV-B radiation exposure

The aim of this study was to investigate the modifying influence of moderate ultraviolet-B (UV-B) radiation exposure on structurally different flavonol glycosides and hydroxycinnamic acid derivatives during pre-harvest using kale, a leafy Brassica species with a wide spectrum of different non-acylated and acylated flavonol glycosides. Juvenile kale plants were treated with short-term (1 day), moderate UV-B radiation [0.22-0.88 kJ m?2 day?1 biologically effective UV-B (UV-B(BE))]. Twenty compounds were quantified, revealing a structure-specific response of flavonol glycosides and hydroxycinnamic acid derivatives to UV-B radiation. A dose- and structure-dependent response of the investigated phenolic compounds to additional UV-B radiation was found. The investigated quercetin glycosides decreased under UV-B; for kaempferol glycosides, however, the amount of sugar moieties and the flavonol glycoside hydoxycinnamic acid residue influenced the response to UV-B. Monoacylated kaempferol tetraglucosides decreased in the investigated UV-B range, whereas the monoacylated kaempferol diglucosides increased strongly with doses of 0.88 kJ m?2 day?1 UV-B(BE) . The UV-B-induced increase in monoacylated kaempferol triglucosides was dependent on the acylation pattern. Furthermore, the hydroxycinnamic acid glycosides disinapoyl-gentiobiose and sinapoyl-feruloyl-gentiobiose were enhanced in a dose-dependent manner under UV-B. While UV-B radiation treatments often focus on flavonol aglycones or total flavonols, our investigations were extended to structurally different non-acylated and acylated glycosides of quercetin and kaempferol.     

UV-B induction of flavonoid biosynthesis in Scots pine (Pinus sylvestris L . ) seedlings

 Cultivation of Scots pine (Pinus sylvestris L.) seedlings under simulated global radiation including the UV-B band (280 – 320 nm; 220 mW m^–2 UV-B_BE) led to increased formation of the diacylated flavonol glucosides 3″,6″-di-p-coumaroyl-astragalin and 3″,6″-di-p-coumaroyl-isoquercitrin in primary and cotyledonary needles, respectively. 3″,6″-Di-p-coumaroyl-astragalin was also the main constitutive diacylated flavonol glucoside in both needle types. This compound predominantly accumulated in primary needles upon UV-B irradiation, and reached concentrations of 2.4 μmol g^–1 fresh weight (fw). Its concentration was only weakly affected in cotyledonary needles. 3″,6″-Di-p-coumaroyl-isoquercitrin was mainly induced in cotyledonary needles with maximum concentrations of 0.8 to 0.9 μmol g^–1 fw, but was virtually unaffected in primary needles under the same irradiation conditions. Pulse labelling with L-(U-¹⁴C)phenylalanine revealed that these metabolites were formed de novo. Phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone synthase (EC 2.3.1.74) were only slightly induced by the UV-B treatment. The results described here represent the first report on UV-B-induced flavonoid biosynthesis in a conifer species. Received: 5 December 1995 / Accepted: 20 March 1996     

Kaempferol Glycosides in the Seed-Coat of Ophiopogon jaburan (Kunth) Lodd.

Blue seed-coats of Ophiopogon jaburan have been found to contain kaempferol, kaempferol 3-glucoside (astragalin), two new glucosides of kaempferol, and a trace amount of an unknown flavonol-like compound. One of the new glucosides was determined to be kaempferol 4′-glucoside and the other to be kaempferol 3, 4′-diglucoside by means of paper-chro-matographic and spectral analyses.     

Direct and indirect effects of solar ultraviolet-B radiation on long-term decomposition

As a result of stratospheric ozone depletion, more solar ultraviolet-B radiation (UV-B, 280–315 nm) is reaching the Earth's surface. Enhanced levels of UV-B may, in turn, alter ecosystem processes such as decomposition. Solar UV-B radiation could affect decomposition both indirectly, by changes in the chemical composition of leaves during growth, or directly by photochemical breakdown of litter and through changes in decomposer communities exposed to sunlight. In this experiment, we studied indirect and direct effects of solar UV-B radiation on decomposition of barley (Hordeum vulgare). We used barley straw and leaf litter grown under reduced UV-B (20% of ambient UV-B) or under near-ambient UV-B (90% of ambient UV-B) in Buenos Aires, Argentina, and decomposed the litter under reduced or near-ambient solar UV-B for 29 months in Tierra del Fuego, Argentina. We found that the UV-B treatment applied during growth decreased the decay rate. On the other hand, there was a marginally significant direct effect of elevated UV-B during the early stages of decomposition, suggesting increased mass loss. The effect of UV-B during growth on decomposition was likely the result of changes in plant litter chemical composition. Near-ambient UV-B received during plant growth decreased the concentrations of nitrogen, soluble carbohydrates, and N/P ratio, and increased the concentrations of phosphorus, cellulose, UV-B-absorbing compounds, and lignin/N ratio. Thus, solar UV-B radiation affects the decomposition of barley litter directly and indirectly, and indirect effects are persistent for the whole decomposition period.     

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.     

Catabolism of flavonol glucosides in plant cell suspension cultures

Catabolism of flavonol glucosides was investigated in plant cell suspension cultures using kaempferol 3-O-β-d-glucoside and kaempferol 7-O-β-d-glucoside labelled with ¹⁴C either in the glucose or in the flavonol moiety. Catabolic rates of glucosides were compared with those of free glucose and kaempferol. All substrates were degraded efficiently by cell cultures of mungbean, soybean, garbanzo bean and parsley. Based on ¹⁴CO₂-formation, glucose from position 3 of kaempferol is 3–5 times more rapidly metabolized than that from position 7. The flavonol nucleus from both isomers is, however, oxidized to the same extent with a considerable portion of the flavonol being incorporated into insoluble polymeric cell material.     

Solar ultraviolet-B radiation influence on Sphagnum bog and Carex fen ecosystems: first field season findings in Tierra del Fuego, Argentina

Stratospheric ozone depletion occurs over Tierra del Fuego, southern Argentina and Chile, in the austral spring and summer due to the precession of the Antarctic ‘ozone hole’ and the general erosion of the ozone layer. Plots receiving either near-ambient or reduced UV-B radiation were established using different louvered plastic film filters over Sphagnum bog and Carex fen ecosystems in October 1996. In the Sphagnum bog system, growth measurements during the late spring and summer showed no significant differences in the moss Sphagnum magellanicum, or the vascular plants (Empetrum rubrum, Nothofagus antarctica, and Tetroncium magellanicum) between near-ambient and attenuated UV-B radiation treatments. In the Carex fen system, leaf length and spike height did not differ in the two dominant species, Carex decidua and C. curta, between UV-B radiation treatments. The length of individual spikelets of C. curta under near-ambient UV-B radiation was less than under the reduced UV-B radiation treatment, but this was not evident in C. decidua. No differences in seed number, seed mass, or viability were seen in either Carex species between the UV-B treatments. Two important constituents of the microfauna that inhabit the Sphagnum bog are testate amoebae and rotifers. These both appeared to be more numerous under near-ambient UV-B radiation than under reduced UV-B radiation. The subtle responses of the Sphagnum and Carex ecosystems may become more apparent in subsequent years as the treatments are continued. Trophic-level changes, such as the differences in number of amoebae and rotifers, may be more sensitive to solar UV-B radiation than growth and productivity of the vegetation.     

Flavonoid constituents of Ephedra alata

Two new flavonol glucosides have been identified in Ephedra alata, namely, herbacetin 8-methyl ether 3-O- glucoside-7-O-rutinoside and herbacetin 7-O-(6″-quinylglucoside). The known flavonoids vicenin II, lucenin III, kaempferol 3-rhamnoside, quercetin 3-rhamnoside and herbacetin 7-glucoside were also found. The structure of the isolated compounds was determined mostly by FABMS and ¹H NMR spectroscopy. The final structure of the new compounds and of herbacetin 7-glucoside was confirmed by ¹³C NMR spectroscopy.     

Flavonoids from Eustoma grandiflorum flower petals

The major flavonoids responsible for flower colours of Eustoma grandiflorum were characterized by TLC, HPLC, spectral and chemical analyses. Anthocyanins were delphinidin 3-rhamnosylgalactoside-5-glucoside and delphinidin 3-galactoside-5-glucoside, each acylated with p-coumaric acid, from the purple cultivar ‘Murasaki no Homare’ and the pelargonidin analogues, each acylated with either p-coumaric or ferulic acids, from the pink cultivar ‘Momo no Mine’. The major flavonol copigments were the 3-rhamnosylgalactoside-7-rhamnoside of myricetin, kaempferol and isorhamnetin and the 3-rhamnosylglucoside-7-rhamnoside of kaempferol and isorhamnetin. Flavonols present acylated with p-coumaric acid were myricetin 3-rhamnosylgalactoside-7-rhamnoside and robinin in both cis and trans forms, and isorhamnetin 3-rhamnosylgalactoside-7-rhamnoside. Robinin also was present acylated with caffeic or ferulic acids. Simulated in vitro colours obtained from the flavonoids present in this germplasm indicated that good blue colours were not attainable. Good blue colours were formed with delphinidin 3-p-coumaroyl-rhamnosylgalactoside-5-glucoside and C-glycosylflavone copigments such as swertisin and isoorientin. These copigments are readily available in other members of the Gentianaceae and this suggests the possibility of genetical engineering endeavours for increasing the colour range of this important new ornamental plant.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

Photoprotection, photosynthesis and growth of tropical tree seedlings under near-ambient and strongly reduced solar ultraviolet-B radiation

Seedlings of two late-successional tropical rainforest tree species, Tetragastris panamensis (Engler) O. Kuntze and Calophyllum longifolium (Willd.), were field grown for 3-4 months at an open site near Panama City (9 degrees N), Panama, under plastic films that either transmitted or excluded most solar UV-B radiation. Experiments were designed to test whether leaves developing under bright sunlight with strongly reduced UV-B are capable of acclimating to near-ambient UV-B conditions. Leaves of T. panamensis that developed under near-ambient UV-B contained higher amounts of UV-absorbing substances than leaves of seedlings grown under reduced UV-B. Photosynthetic pigment composition, content of alpha-tocopherol, CO(2) assimilation, potential photosystem II (PSII) efficiency (evaluated by F(v)/F(m) ratios) and growth of T. panamensis and C. longifolium did not differ between seedlings developed under near-ambient and reduced solar UV-B. When seedlings were transferred from the reduced UV-B treatment to the near-ambient UV-B treatment, a pronounced inhibition of photosynthetic capacity was observed initially in both species. UV-B-mediated inhibition of photosynthetic capacity nearly fully recovered within 1 week of the transfer in C. longifolium, whereas in T. panamensis an about 35% reduced capacity of CO(2) uptake was maintained. A marked increase in UV-absorbing substances was observed in foliage of transferred T. panamensis seedlings. Both species exhibited enhanced mid-day photoinhibition of PSII immediately after being transferred from the reduced UV-B to the near-ambient UV-B treatment. This effect was fully reversible within 1d in T. panamensis and within a few days in C. longifolium. The data show that leaves of these tropical tree seedlings, when developing in full-spectrum sunlight, are effectively protected against high solar UV-B radiation. In contrast, leaves developing under conditions of low UV-B lacked sufficient UV protection. They experienced a decline in photosynthetic competence when suddenly exposed to near-ambient UV-B levels, but exhibited pronounced acclimative responses.     

Flavonoids of Mayaca fluviatilis (Mayacaceae)

Mayaca is an aquatic monocot of the monogeneric family Mayacaceae. The flavonol glycosides quercetin 3-O-glucoside, quercetin 3-O-rutinoside, and kaempferol 3-O-glucoside, and the flavone luteolin 5-O-glucoside were found in methanolic leaf extracts. The presence of flavonol and flavone O-glycosides sets the Mayacaceae apart from the Commelinaceae, which accumulates predominantly flavone C-glycosides.     

Differential stimulation of an arid-environment winter ephemeral Dimorphotheca pluvialis (L.) Moench by ultraviolet-B radiation under nutrient limitation

A South African winter ephemeral D. pluvialis was exposed, under low and high nutrient conditions, to four different daily doses of biologically effective UV-B radiation. These simulated different depletions (range 0–30%) in the ozone layer at the southerly distribution limit (33° 56′S) of this species, and included daily UV-B doses received at the northerly distribution limit (26° 38′S) without ozone depletion. Growth inhibition by increased UV-B radiation was observed during early vegetative stages, but only under low nutrient conditions. Thereafter, net CO₂ assimilation rate, growth and reproduction were stimulated by an increase in UV-B radiation, though doses above those approximating a 20% ozone depletion appeared to be inhibitory. Differential stimulation occurred in the two nutrient treatments. Under high nutrient conditions, photosynthesis (specifically carboxylation efficiency), and numbers of leaves, inflorescences and diaspores per plant, and leaf areas increased, but leaf thickness decreased with increased UV-B radiation. Under low nutrient conditions, dry masses of leaves, stems, inflorescences and diaspores, and total above-ground dry masses increased with increased UV-B radiation. Foliar organic carbon and nitrogen concentrations and foliar concentrations of UV-B absorbing compounds were unaffected by increased UV-B radiation, but foliar P concentrations declined. Diaspore viability declined with increased UV-B radiation. The net effect was a 35 to 43% reduction in viable diaspore production under high nutrient conditions at UV-B doses equivalent to those currently received at the northerly distribution limit during the reproductive phase. It is concluded that anticipated increases in UV-B radiation could reduce regeneration success, and seedling survival in areas of low soil fertility, particularly at lower latitudes, and consequently increase the risk of localized population extinctions from stochastic causes.     

Acylated flavonol diglucosides from Lotus polyphyllos

Three acylated flavonol diglucosides, kaempferol 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quercetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.     

Exposure to solar UV-B radiation accelerates mass and lignin loss of <Emphasis Type="Italic">Larrea tridentata</Emphasis> litter in the Sonoran Desert

We assessed whether exposure to solar ultraviolet-B radiation (UV-B) affects the mass loss of Larrea tridentata (creosotebush) litter in the Sonoran Desert of central Arizona. We placed three types of litter (leaves, twigs, or a natural mixture of leaves, twigs, and seeds) in bags constructed of UV-B-transmitting or UV-B-absorbing filter material that allowed either 85% (near-ambient UV-B treatment) or 15% (reduced UV-B treatment) of the biologically effective solar UV-B to reach litter inside the bags. Bags were placed outdoors for 4–5 months during the winter at two sites: a balcony or on the soil surface of the desert. Mass loss of leaf litter was greater under near-ambient UV-B than reduced UV-B at both sites: 21 (near-ambient) vs. 18% (reduced) on the balcony, and 18 vs. 14% at the desert site. Mass loss of twig litter was also greater under near-ambient UV-B at the desert site. Mass loss of the natural mixture of litter was also greater when exposed to near-ambient UV-B on the balcony, and tended to be greater at the desert site. We estimate that about 14–22% of the total mass loss of leaf litter during our 4–5 month experiments was attributable to solar UV-B exposure. Leaf litter exposed to near-ambient UV-B had lower concentrations of lignin, and fats and lipids, and slightly higher concentrations of holocellulose. The greater mass loss of litter under near-ambient UV-B appeared mainly attributable to loss of lignin, although losses of fats and lipids were also appreciable. A primary reason for greater mass loss of litter under solar UV-B appeared to be photodegradation, particularly of lignin.     

The Role of Flavonol Glycosides and Carotenoids in Protecting Soybean from Ultraviolet-B Damage

The increase in ultraviolet-B (UV-B; 0.290-0.320 [mu]m) radiation received by plants due to stratospheric ozone depletion heightens the importance of understanding UV-B tolerance. Photosynthetic tissue is believed to be protected from UV-B radiation by UV-B-absorbing compounds (e.g. flavonoids). Although synthesis of flavonoids is induced by UV-B radiation, its protective role on photosynthetic pigments has not been clearly demonstrated. This results in part from the design of UV-B experiments in which experimental UV-A irradiance has not been carefully controlled, since blue/UV-A radiation is involved in the biosynthesis of the photosynthetic pigments. The relationship of flavonoids to photosynthetic performance, photosynthetic pigments, and growth measures was examined in an experiment where UV-A control groups were included at two biologically effective daily UV-B irradiances, 14.1 and 10.7 kJ m-2. Normal, chlorophyll-deficient, and flavonoid-deficient pigment isolines of two soybean (Glycine max) cultivars that produced different flavonol glycosides (Harosoy produced kaempferol, Clark produced quercetin and kaempferol) were examined. Plants with higher levels of total flavonoids, not specific flavonol glycosides, were more UV-B tolerant as determined by growth, pigment, and gas-exchange variables. Regression analyses indicated no direct relationship between photosynthesis and leaf levels of UV-B-absorbing compounds. UV-B radiation increased photosynthetic pigment content, along with UV-B-absorbing compounds, but only the former (especially carotenoids) was related to total biomass (r2 = 0.61, linear) and to photosynthetic efficiency (negative, exponential relationship, r2 = 0.82). A reduction in photosynthesis was associated primarily with a stomatal limitation rather than photosystem II damage. This study suggests that both carotenoids and flavonoids may be involved in plant UV-B photoprotection, but only carotenoids are directly linked to photoprotection of photosynthetic function. These results additionally show the importance of UV-A control in UV-B experiments conducted using artificial lamps and filters.