Chemical Profiling and Biological Activity of Extracts from Nine Norwegian Medicinal and Aromatic Plants.

There is an increased interest in identifying beneficial compounds of plant origin that can be added to animal diets to improve animal performance and have a health-promoting effect. In the present study, nine herb species of the Norwegian wild flora or which can be cultivated in Norway were selected for phytogenic evaluation (hops, maral root, mint, oregano, purslane, rosemary, roseroot, sweet wormwood, yarrow). Dried herbs were sequentially extracted with dichloromethane (DCM), ethanol (EtOH) and finally water (H2O) by ultrasound-assisted extraction (UAE). The UAE protocol was found to be more rational than conventional Soxhlet with respect to DCM extraction. Total extraction yield was found to be highest for oregano (Origanum vulgare) with 34.4 g 100-1 g dry matter (DM). H2O-extracts gave the highest yields of the three solvents, with up to 25 g 100-1 g DM for purslane (Portulaca oleracea ssp. sativa) and mint (Mentha piperita). EtOH- and H2O-extracts were the most efficient extracts with respect to free radical scavenging capacity (ABTS (=2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and oregano, mint, hops (Humulus lupulus) and maral root-leaves (Leuzea carthamoides) were found to be the most efficient antioxidant sources. Hops (EtOH-extract) contained α- and ß-acids, xanthohumols, chlorogenic acid and the hitherto unreported 3-O-glucosides of kaempferol and quercetin. Maral root-leaves contained among other compounds hexosides of the 6-hydroxy- and 6-methoxy-kaempferol and -quercetin, whereas roseroot (Rosea rhodiola) revealed contents of rosavin, rhodiosin and rhodionin. Sweet wormwood (Artemisia annua) contained chlorogenic acid and several derivatives thereof, scopoletin and poly-methylated flavones (eupatin, casticin, chrysoplenetin). Antimicrobial potential of different plant extracts was demonstrated against Gram-positive and Gram-negative bacteria using the indicator organisms Staphylococcus aureus, and Escherichia coli, and the Atlantic salmon bacterial pathogens Moritella viscosa, Tenacibaculum finnmarkense and Aliivibrio wodanis. DCM extracts possessed the highest activities. Data demonstrate the potential ability of herb extracts as natural antimicrobials. However, future safety studies should be performed to elucidate any compromising effect on fish health.

A novel bicyclic lactone and other polyphenols from the commercially important vegetable Anthriscus cerefolium.

Garden chervil, Anthriscus cerefolium (L.) Hoffm. is an important herb commonly applied in Norwegian large-scale commercial kitchens. This species is a highly enriched source of phenolics, containing 1260 mg gallic acid equivalents (GAE) 100-1 g DM, however, the individual phenolic compounds have been scarcely characterized. Here we report on the qualitative and quantitative content of phenolics in garden chervil. The structure of the main phenolic compound was elucidated to be the previously undescribed compound 1,3-dicaffeoyl-5-malonyl-δ-quinide (1) by means of 1D- and 2D NMR and high-resolution mass spectrometry. The known flavones apigenin 7-O-ß-(2″-apiofuranosylglucopyranoside) (= apiin) (2), apigenin 7-(2″-apiosyl-6″-malonylglucoside) (3) and luteolin 7-glucoside (4) were also identified. Compound 3 is reported for the first time from this plant species. The main phenolic compound, 1,3-dicaffeoyl-5-malonyl-δ-quinide, exhibited moderate cytotoxicity towards acute monocytic leukaemia cells (MOLM-13) and rat kidney epithelial cells (NRK) with EC50 between 400 and 600 µM.

Decoupling of Plant Growth and Accumulation of Biologically Active Compounds in Leaves, Roots, and Root Exudates of Hypericum perforatum L. by the Combination of Jasmonate and Far-Red Lighting.

The plant hormone jasmonic acid (JA) fine tunes the growth-defense dilemma by inhibiting plant growth and stimulating the accumulation of secondary compounds. We investigated the interactions between JA and phytochrome B signaling on growth and the accumulation of selected secondary metabolites in Hypericum perforatum L., a medically important plant, by spraying plants with methyl jasmonate (MeJA) and by adding far-red (FR) lighting. MeJA inhibited plant growth, decreased fructose concentration, and enhanced the accumulation of most secondary metabolites. FR enhanced plant growth and starch accumulation and did not decrease the accumulation of most secondary metabolites. MeJA and FR acted mostly independently with no observable interactions on plant growth or secondary metabolite levels. The accumulation of different compounds (e.g., hypericin, flavonols, flavan-3-ols, and phenolic acid) in shoots, roots, and root exudates showed different responses to the two treatments. These findings indicate that the relationship between growth and secondary compound accumulation is specific and depends on the classes of compounds and/or their organ location. The combined application of MeJA and FR enhanced the accumulation of most secondary compounds without compromising plant growth. Thus, the negative correlations between biomass and the content of secondary compounds predicted by the growth-defense dilemma were overcome.

Flavonoids and other phenolics in herbs commonly used in Norwegian commercial kitchens.

Significant quantities of several important herbs are processed and consumed from Norwegian commercial kitchens annually although surprisingly the contents of polyphenols have been scarcely characterized. We here report on the qualitative and quantitative content of polyphenolic compounds from ten of the most utilized herbs. From parsley (Petroselinum crispum) var. Darki, isorhamnetin 3-(6″-malonylglucoside)-7-glucoside (2) and diosmetin 7-(2″-apiosyl-6″-malonylglucoside) (8) are reported for the first time, in addition to seven known flavonoids, some of which are reported for the first time from this plant species. Oregano, rosemary and thyme contained the highest amounts of total phenolics with maximum levels of 23.8, 24.2 and 23.4 mg GAE g-1 dry matter, respectively. Fresh herbs contained significantly higher quantities of phenolics than processed, dried herbs. Parsley, coriander, dill and thyme were the richest sources of flavonoids among the investigated herbs.

Stress acclimation and particulate matter accumulation in Pinus sylvestris saplings affected by moderate combinations of urban stressors.

To predict how the function of urban vegetation and the provision of ecosystem services respond to combinations of natural and anthropogenic drivers, a better understanding of multiple stress interactions is required. This study tested combined effects of moderate levels of drought, soil salinity and exposure to diesel exhaust on parameters of physiology, metabolism, morphology and growth of Pinus sylvestris L. saplings. We found that plant responses were primarily dominated by single stressors and a few two-way interactions. Stressor combinations did not have considerable additional negative effects on plant performance compared to single stressors. Hence, synergistic and antagonistic interactions were rare and additive effects frequent. Drought cycles caused most negative effects, from chlorophyll a fluorescence and epicuticular wax content to growth responses, while soil salinity caused fewer negative effects but contributed to reduction in fine root growth and fluorescence parameters at low air contamination. Interestingly, the air contamination alone had only marginal effects on plant morphology and growth, but contributed an antagonistic effect, dampening the negative effect of drought and salinity on the maximum quantum efficiency of PSII photochemistry (Fv/Fm) and fine root biomass. Although, these effects were moderate, it appears that exhaust exposure had a cross-acclimation effect on plant responses to drought and salinity. We also found that salinity had a negative effect on the accumulation of particulate matter on shoots, illustrating that the plant stress situation can affect the provisioning of certain ecosystem services like pollution attenuation. These findings have implications for the understanding of the complex natural and anthropogenic stress situation of urban, and how to maintain the ecological functions and delivery of ecosystem services.

From producer to consumer: greenhouse tomato quality as affected by variety, maturity stage at harvest, transport conditions, and supermarket storage.

Possible causes for differences in quality traits at the time of buying were studied in two widely different red tomato types. Three maturity stages were harvested from commercial greenhouses and transferred immediately to controlled environments simulating different storage, transport, and supermarket conditions. Results show significant differences in development of color, fruit firmness, contents of soluble solids (SSC), titratable acids (TTA), phenolics, and carotenoids from harvest to sale, as related to postharvest conditions. Fruit firmness, SSC, and TTA of vine-ripened red cherry tomatoes was 30, 55 and 11% higher than for those harvested at breakers and ripened to red. Temperature, light, UVC radiation, or ethylene during 4 days transport affected tomato quality traits, and differences persisted during 3 weeks of supermarket storage. Ethylene exposure gave a 3.7-fold increase in lycopene content in cherry tomatoes, whereas UVC hormesis revealed a 6-fold increase compared with the control. Results can be used to update recommendations concerning optimal handling.

Black chokeberry juice (Aronia melanocarpa) reduces incidences of urinary tract infection among nursing home residents in the long term--a pilot study.

Urinary tract infection (UTI) is a major problem in nursing homes, and the mainstay of treatment is antibiotics. The increasing prevalence of uropathogens resistant to antimicrobial agents has stimulated interest in specific nutrients, for example, cranberries, to prevent recurring UTI. Black chokeberry (Aronia melanocarpa [Michx] Elliott) is a rich source of phenolics, and thus, dietary intake of black chokeberry juice may reduce the incidences of UTI requiring medical treatment. In this pilot study, we examined the frequency of medically treated UTI among residents in 6 nursing homes who were offered black chokeberry juice or a placebo during a 6-month crossover intervention. The residents were offered a placebo drink for a 3-month period followed by chokeberry juice for the next 3 months (group A) or vice versa (group B). The juice was characterized by a high content of total phenolics (715 mg gallic acid equivalent, 100 mL(-1)), including B-type procyanidins, anthocyanins, and chlorogenic acids. Daily intake of chokeberry juice was 156 mL per resident in group A (n = 110) and 89 mL per resident in group B (n = 126). Urinary tract infection comprised 55% of all medically treated infections during the study period. The results revealed no immediate reduction in the frequency of UTI or the total use of antibiotics; however, during the subsequent 3-month period of juice administration, a reduction in antibiotics toward UTI was observed in both groups. The incidence of UTI was reduced by 55% in group A and 38% in group B. No changes in other infections or in use of prophylactics were observed.

Immunomodulating activity of Aronia melanocarpa polyphenols.

The immunomodulating effects of isolated proanthocyanidin-rich fractions, procyanidins C1, B5 and B2 and anthocyanins of Aronia melanocarpa were investigated. In this work, the complement-modulating activities, the inhibitory activities on nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages and effects on cell viability of these polyphenols were studied. Several of the proanthocyanidin-rich fractions, the procyanidins C1, B5 and B2 and the cyanidin aglycone possessed strong complement-fixing activities. Cyanidin 3-glucoside possessed stronger activity than the other anthocyanins. Procyanidins C1, B5 and B2 and proanthocyanidin-rich fractions having an average degree of polymerization (PD) of 7 and 34 showed inhibitory activities on NO production in LPS-stimulated RAW 264.7 mouse macrophages. All, except for the fraction containing proanthocyanidins with PD 34, showed inhibitory effects without affecting cell viability. This study suggests that polyphenolic compounds of A. melanocarpa may have beneficial effects as immunomodulators and anti-inflammatory agents.

Effects of Aronia melanocarpa constituents on biofilm formation of Escherichia coli and Bacillus cereus.

Many bacteria growing on surfaces form biofilms. Adaptive and genetic changes of the microorganisms in this structure make them resistant to antimicrobial agents. Biofilm-forming organisms on medical devices can pose serious threats to human health. Thus, there is a need for novel prevention and treatment strategies. This study aimed to evaluate the ability of Aronia melanocarpa extracts, subfractions and compounds to prevent biofilm formation and to inhibit bacterial growth of Escherichia coli and Bacillus cereus in vitro. It was found that several aronia substances possessed anti-biofilm activity, however, they were not toxic to the species screened. This non-toxic inhibition may confer a lower potential for resistance development compared to conventional antimicrobials.

Extracts, anthocyanins and procyanidins from Aronia melanocarpa as radical scavengers and enzyme inhibitors.

Extracts, subfractions, isolated anthocyanins and isolated procyanidins B2, B5 and C1 from the berries and bark of Aronia melanocarpa were investigated for their antioxidant and enzyme inhibitory activities. Four different bioassays were used, namely scavenging of the diphenylpicrylhydrazyl (DPPH) radical, inhibition of 15-lipoxygenase (15-LO), inhibition of xanthine oxidase (XO) and inhibition of α-glucosidase. Among the anthocyanins, cyanidin 3-arabinoside possessed the strongest and cyanidin 3-xyloside the weakest radical scavenging and enzyme inhibitory activity. These effects seem to be influenced by the sugar units linked to the anthocyanidin. Subfractions enriched in procyanidins were found to be potent α-glucosidase inhibitors; they possessed high radical scavenging properties, strong inhibitory activity towards 15-LO and moderate inhibitory activity towards XO. Trimeric procyanidin C1 showed higher activity in the biological assays compared to the dimeric procyanidins B2 and B5. This study suggests that different polyphenolic compounds of A. melanocarpa can have beneficial effects in reducing blood glucose levels due to inhibition of α-glucosidase and may have a potential to alleviate oxidative stress.

In vitro inhibition of cytochrome P450 3A4 by Aronia melanocarpa constituents.

Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin.

Indications of heightened constitutive or primed host response affecting the lignin pathway transcripts and phenolics in mature Norway spruce clones.

Two mature clones of Norway spruce (Picea abies (L.) Karst.) that have previously been shown to have differential degrees of resistance towards the necrotrophic pathogen Heterobasidion parviporum (Niemelä & Korhonen) were compared with respect to the primed defense expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds from one year to the next. The host's response to physical wounding and pathogen inoculation was examined in the initial year, whereas indications of heightened basal defense level or primed response, and responses to re-wounding, were examined the following year. The responses of the two clones to wounding and pathogen inoculation, examined in the initial year, differed; the increases in lignin and phenolics were more distinct in response to the pathogen than to wounding alone. The more resistant clone 589 had higher initial lignin concentrations in the cell walls when compared with clone 409, and these remained higher in clone 589 over both years and increased after the treatments. Both clones responded at the transcriptional and chemical levels to wounding; changes were evident both in the initial wounds and when re-wounded the following year. There were distinct differences in the basal transcript levels of the lignin pathway-related genes, phenolics and total lignin levels in healthy tissue from the initial year to the following year indicative of a primed host response or at least altered constitutive level of defense expression.

Influence of repeated short-term nitrogen limitations on leaf phenolics metabolism in tomato.

High concentrations of phenolics have been shown to play a role in plant resistance to pathogens. One way to obtain increased phenolic concentrations in plant tissues is to limit mineral nitrogen (N) availability; however, over long periods, this treatment will have a negative effect on plant growth. The aim of our study was to determine the effect of repeated short-term N limitations on plant growth and phenolic metabolism in leaves. Tomato plants (Solanum lycopersicum, cv. Pixie) were subjected to two successive 10-day N-limitation periods (0.15 mM NO(3)(-), 0.01 mM NH(4)(+)), followed by periods of full nutrient supply (15 mM NO(3)(-), 1.2 mM NH(4)(+)). Additionally, other plants were subjected to either of these two limitation periods, and a set of control plants was given a full nutrient supply during the entire period. The phenolic metabolism was monitored by measuring the leaf concentrations of chlorogenic acid, three flavonol glycosides (quercetin and kaempferol derivatives) and two major anthocyanins, together with the expression of eight structural genes and three transcription factors of the phenylpropanoid pathway. The relative growth rate of the plants decreased during the N-limitation periods but was restored as soon as N was resupplied. Each N-limitation period resulted in an up-regulation of the phenolic biosynthetic pathway, as demonstrated by an increase in the leaf phenolic concentration and an up-regulation of the related genes. The genes in the phenolic pathway were down-regulated immediately when N was resupplied; however, the leaf concentrations of several phenolics, particularly flavonol glycosides, were maintained at significantly higher levels than in the control plants for up to 17 days after the end of the first limitation. The amplitude of the increase in leaf phenolic concentration did not depend on the number of N-limitation periods to which the plant was subjected, which indicates that the plants did not acclimate to nitrogen limitation. Successive N-limitation periods resulted in additive increases in flavonol glycoside concentrations.

Properties of chalconaringenin and rutin isolated from cherry tomatoes.

Fresh cherry tomatoes cv. 'Susanne' contain more of the two flavonoids chalconaringenin (CN) and rutin than lycopene. Therefore some properties including antioxidant behavior of the flavonoids were studied. The two flavonoids were extracted from peel and isolated by use of different chromatographic methods. Molecular absorbtivities were found to be 26907 for CN and 20328 abs M(-1) cm(-1) for rutin. Both compounds exhibited properties as antioxidants through several assays, and rutin was found to be the strongest antioxidant except in one assay. None of the assays revealed pro-oxidative effects. As naringenin rather than CN is frequently reported as a tomato constituent, the stability of CN was investigated in order to detect potential ways of isomerization during sample preparation. CN isomerized slowly both under UVB radiation and in alkaline solutions. Thus, such factors do not explain the occurrence of naringenin in tomato samples. The deficiency in reports on CN may be explained by the similarity in chromatographic behaviors of CN and naringenin, and due to the fact that they have same molecular weights.

Norwegian-grown Jerusalem artichoke (Helianthus tuberosus L.): morphology and content of sugars and fructo-oligosaccharides in stems and tubers.

BACKGROUND: Two field trials growing Jerusalem artichoke (Helianthus tuberosus L.) are discussed. Twenty genetic variants were compared in order to find genotypes suitable for Norwegian production. A second trial investigated the above-ground part of early and late variants. RESULTS: The highest yield (28.7 t ha(-1)) and highest amount of tubers per plant was obtained in early variants, e.g. the white Tysnes gave 1.72 kg tubers plant(-1). Early variants had a markedly lower portion of smooth tubers. Late variants gave the most preferable tuber shape, but low yield. Only a weak correlation was found between dry matter content and total content of fructo-oligosaccharides (FOS) (r = 0.255) in the tubers. The mean content of FOS including sucrose through all variants was found to be 116 g kg(-1) fresh weight (FW) or 550 g kg(-1) dry weight (DW). The average chain length of FOS in the tubers was found to be DP3.9. There was no difference in tuber FOS content between early and late variants. The highest above-ground amount biomass was found for the late variants when harvested in September. The content of soluble carbohydrates was found to be highest in stalks in August (sucrose and FOS major compounds). CONCLUSION: Early variants give the highest tuber yield under Norwegian growing conditions. Late variants give highest above-ground biomass.

Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum.

BACKGROUND: Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism. RESULTS: The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins. CONCLUSIONS: CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism.

Synergetic effects of nitrogen depletion, temperature, and light on the content of phenolic compounds and gene expression in leaves of tomato.

Tomato plants (Solanum lycopersicum, cv. Suzanne) were subjected to complete nutrient solution or a solution without nitrogen (N), and placed at different temperatures and light conditions to test the effects of environment on flavonoids and caffeoyl derivatives and related gene expression. N depletion during 4-8days resulted in enhanced levels of flavonoids and caffeoyl derivatives. Anthocyanins showed pronounced increased levels when lowering the growth temperature from 24 degrees C to 18 degrees C or 12 degrees C. Flavonol levels increased when the light intensity was increased from 100 micromol m(-2) s(-1) PAR to 200 micromol m(-2) s(-1) PAR. Synergistic effects of the various environmental factors were observed. The increase in content of quercetin derivatives in response to low temperatures was only found under conditions of N depletion, and especially at the higher light intensity. Expression of structural genes in the phenylpropanoid and flavonoid pathways, PAL (phenylalanine ammonia lyase), CHS (chalcone synthase), F3H (flavanone 3-hydroxylase), and FLS (flavonol synthase) increased in response to N depletion, in agreement with a corresponding increase in flavonoid and caffeoyl content. Expression of these structural genes generally also increased in response to lower temperatures. As indicated through expression studies and correlation analysis, effects of N depletion were apparently mediated through the overall regulators of the pathway the MYB transcription factor ANT1 (ANTHOCYANIN 1) and SlJAF13 (a bHLH transcription factor orthologue of petunia JAF13 and maize RED genes). A PAL gene (PAL6) was identified, and correlation analysis was compatible with PAL6 being an actively expressed gene with function in flavonoid synthesis.

Analysis of phenolic compounds in six Norwegian plum cultivars (Prunus domestica L.).

Six European plum cultivars ( Prunus domestica L.) grown in Norway have been studied with respect to phenolic composition. Neochlorogenic acid was found to be the most important phenolic acid in all cultivars. Together with other phenolic acids, this compound varied significantly in amount among the cultivars. Cyanidin 3-rutinoside was found to account for >60% of the total anthocyanin content. Minor amounts of flavonols (rutin and quercetin 3-glucoside) were detected in all cultivars. Total antioxidant capacity varied from 814 to 290 micromol of Trolox 100 g(-1) of fresh weight. Measurement of total phenolic content in terms of Prussian blue complex formation revealed a method failure of magnitude order compared to results obtained by HPLC. Comparison of the response factors of a range of phenolic compounds obtained upon analysis by the Prussian blue and Folin-Ciocalteu assays revealed that the latter method returned higher yields in terms of gallic acid (GAE).

The endogenous GL3, but not EGL3, gene is necessary for anthocyanin accumulation as induced by nitrogen depletion in Arabidopsis rosette stage leaves.

The bHLH transcription factors EGL3 (ENHANCER OF GLABRA3) and its close homologue GL3 (GLABRA3) are important regulators of the anthocyanin pathway in Arabidopsis thaliana, and together with TTG1 (a WD40 repeat protein) and MYB transcription factors regulate specific genes in the pathway. In response to nitrogen depletion, the MYB genes PAP1/PAP2 (production of anthocyanin pigment 1/2) and GL3 are strongly induced, and anthocyanin synthesis is activated in seedlings and rosette stage plants. In this study we show that anthocyanins accumulate in both wild type and egl3, but not in gl3 loss-of-function mutants when depleted of nitrogen. Several structural genes of flavonoid metabolism including CHS (chalcone synthase), FLS1 (flavonol synthase 1) and ANS (anthocyanidin synthase) were induced in response to nitrogen depletion in wild type as well as in the egl3 and gl3 mutants. Strikingly, in the gl3 mutant DFR (dihydroflavonol-4-reductase) transcript level was only 2% of the levels in wild type or egl3 mutant. Hence, low expression of DFR appears to be the bottleneck preventing anthocyanin synthesis in the gl3 mutant. The specific effect on DFR, but not ANS is compatible with involvement of the MYBL2 inhibitor.

Temperature and nitrogen effects on regulators and products of the flavonoid pathway: experimental and kinetic model studies.

The flavonoid pathway is known to be up-regulated by different environmental stress factors. Down-regulation of the pathway is much less studied and is emphasized in the present work. Flavonoid accumulation was induced by exposing plants for 1 week to nitrogen depletion at 10 degrees C, giving high levels of anthocyanins and 3-glucoside-7-rhamnosides, 3,7-di-rhamnosides and 3-rutinoside-7-rhamnosides of kaempferol and quercetin. Flavonol accumulation as influenced by temperatures and nitrogen supply was not related to the glycosylation patterns but to the classification as quercetin and kaempferol. When nitrogen was re-supplied, transcripts for main regulators of the pathway, PAP1/GL3 and PAP2/MYB12, fell to less than 1 and 0.1% of initial values, respectively, during 24 h in the 15-30 degrees C temperature range. Anthocyanins showed a half-life of approximately 1 d, while the degradation of flavonols was much slower. Interestingly, the initial fluxes of anthocyanin and flavonol degradations were found to be temperature-independent. A kinetic model for the flavonoid pathway was constructed. In order to get the observed concentration-temperature profiles as well as the temperature compensation in the flavonoid degradation flux, the model predicts that the flavonoid pathway shows an increased temperature sensitivity at the end of the pathway, where the up-regulation by PAP/GL3 has been found to be largest.