Rune Slimestad

Nitrogen deficiency enhances expression of specific MYB and bHLH transcription factors and accumulation of end products in the flavonoid pathway

Expression of regulators of the flavonoid pathway was examined in Arabidopsisthaliana wild type and pap1D plants, the latter being a T-DNA activation-tagged line over-expressing the PAP1/MYB75 gene which is a positive regulator of the pathway. Anthocyanin accumulation was induced in plants grown in soil, on agar plates, and hydroponics by withdrawing nitrogen from the growth medium. The agar-grown seedlings and rosette stage plants in hydroponics were further explored, and showed that nitrogen deficiency resulted in the accumulation of not only anthocyanins, but also flavonols. The examination of transcript levels showed that the general flavonoid pathway regulators PAP1 and PAP2 were up-regulated in response to nitrogen deficiency in wild type as well as pap1D plants. Interestingly, PAP2 responded much stronger to nitrogen deficiency than PAP1, 200- and 6-fold increase in transcript levels, respectively, for wild-type seedlings. In rosette leaves the increase was 900-fold for PAP2 and 6-fold for PAP1. At least three different bHLH domain transcription factors promote anthocyanin synthesis, and transcripts for one of these, i.e. GL3 were found to be sixfold enhanced by nitrogen deficiency in rosette leaves. The MYB12 transcription factor, known to regulate flavonol synthesis, was slightly induced by nitrogen deficiency in seedlings. In conclusion, four out of eight regulators involved in the flavonoid pathway showed an enhanced expression from 2 to 1,000 times in response to nitrogen deficiency. Together with MYB factors, especially PAP2, GL3 appears to be the BHLH partner for anthocyanin accumulation in response to nitrogen deficiency.

Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the first step in the phenylpropanoid pathway, and is considered an important regulation point between primary and secondary metabolism. In the present work we analyzed expression of the PAL genes in leaves of Arabidopsis thaliana rosette-stage plants in response to nitrogen depletion at temperatures ranging from 5 to 30 degrees C. Only PAL1 and PAL2 responded strongly to both environmental factors, nitrogen and temperature. Regardless of nitrogen treatments, PAL1 and 2 transcript levels increased at 5 and 10 degrees C. Averaged across all temperatures, nitrogen depletion led to a two-fold increase in PAL1 and PAL2 transcripts. PAL activity was correlated with PAL transcript levels (R=0.94). Accumulation of major soluble phenylpropanoids, sinapic acid esters and flavonoids, increased in response to lowering temperature. The flavonoids, kaempferols, quercetins and anthocyanins, showed significantly increased levels as a result of nitrogen depletion (two-, five- and six-fold increases, respectively) when averaged across all temperatures. PAL1, PAL2 and PAL4 have previously been shown to be related with tissue-specific lignin synthesis, and the present work shows that PAL1 and PAL2 also have functional specialization in abiotic environmental-triggered flavonoid synthesis.

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.

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.

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.

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.

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).

Syringetin 3-O-(6″-acetyl)-β-glucopyranoside and other flavonols from needles of norway spruce, Picea abies

Abstract The novel flavonol, syringetin 3- O -(6″-acetyl)- β -glucopyranoside, has been isolated from needles of Norway spruce ( Picea abies ) together with the 3- O -(6″-acetyl)- β -glucopyranosides of isorhamnetin and kaempferol, the 3- O -(6″- α -rhamnopyranosyl)- β -glucopyranosides of laricitrin, isorhamnetin, myricetin, quercetin and kaempferol and the 3- O - β -glucopyranosides of laricitrin, isorhamnetin, myricetin, quercetin and kaempferol. Most of the flavonols have been isolated for the first time from Norway spruce. Kaempferol 3- O -(6″-acetyl)- β -glucopyranoside has previously been isolated from Senecio aureus , but without determination of the binding site of the acetyl group. Structure determination of the flavonols was achieved from TLC, 1 H NMR and UV shift reagent data, and, in most cases, 13 C NMR and MS.

Combination of chromatographic techniques for the preparative isolation of anthocyanins — applied on blackcurrant (Ribes nigrum) fruits

A combination of column chromatography on Toyopearl HW-40F gel and reversed-phase high-performance liquid chromatography enabled us to preparatively separate anthocyanins, without any re-application of overlapping bands of the major (the 3-O-glucosides and 3-O-rutinosides of delphinidin and cyanidin) and minor (the 3-O-rutinosides of peonidin and malvidin) anthocyanins in a 0.81-g sample from blackcurrant (Ribes nigrum) fruits. Anthocyanins substituted with methoxyl groups on the aglycone have never been detected previously in the genus Ribes. By variation of sample loading, flow-rate and solvent strength, Toyopearl HW-40F gel gave rise to chromatograms with higher resolution between the anthocyanidin 3-rutinoside and anthocyanidin 3-glucoside bands than Sephadex LH-20 gel, in all cases.

Anthocyanins from a Norwegian potato cultivar

Abstract The main anthocyanins of purple sprouts of a Norwegian potato cultivar, Solanum tuberosum L., were isolated from a purified methanolic extract by preparative HPLC. Their structures were determined to be the novel anthocyanins, petunidin 3- O -[6- O -(4- O - E -caffeoyl- O -α-rhamnopyranosyl)-β-glucopyranoside]-5- O -β-glucopyranoside ( 1 ) (10%) and peonidin, 3- O -[6- O -(4- O - E -caffeoy1- O -α-rhamnopyranosyl)-β-glucopyranoside]-5- O -β-glucopyranoside ( 2 ) (6%) in addition to petunidin, 3- O -[6- O -(4- O-E -p- coumaroyl -O- α-rhamnopyranosyl)-β-glucopyranoside]-5- O -β-g1ucopyranoside, petanin ( 3 ) (37%) and peonidin, 3- O -[6- O -(4- O - E - p -coumaroyl- O -α-rhamnopyranosyl)-β-glucopyranoside]-5- O -β-glucopyranoside, peonanin ( 4 ) (25%). The same major anthocyanins, however, in other proportions (4, 54, and 32%, for 1 , 3 , and 4 , respectively), were also found in the thin violet zone located in the flesh 0.5–1 cm from the surface of the tubers. This is the first report on anthocyanins from S . tuberosum, which are acylated with caffeic acid.