Induction of hydroxycinnamic acid amides and tryptophan by jasmonic acid, abscisic acid and osmotic stress in barley leaves

The effects of jasmonic acid (JA) and abscisic aid (ABA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. Treatment with JA at 100 microM for 48 h induced accumulation of four compounds in barley primary leaves. The accumulation of these compounds was also observed after treatment with ABA at 100 microM. The induced compounds were identified as p-coumaroylputrescine, p-coumaroylagmatine, p-coumaroyl-3-hydroxyagmatine and tryptophan by spectroscopic methods. The profiles of compounds induced by application of JA and ABA were different. JA exhibited stronger inducing activity for hydroxycinnamic acid amides than ABA, while ABA was more active in tryptophan accumulation. The major hydroxycinnamic acid amides in JA- and ABA-treated leaves were p-coumaroylagmatine and p-coumaroyl-3-hydroxyagmatine, respectively. These differences suggested that JA and ABA act in distinct modes. The induction of these compounds was also observed in leaf segments treated with 1 M sorbitol and glucose. These findings suggested that JA and ABA are involved in accumulation of hydroxycinnamic acid amides and tryptophan in response to osmotic stress in barley.     

Isoflavone Content in Subterranean Clover Germplasm from Sardinia

Subterranean clover (Trifolium subterraneum) is an important pasture legume, and Sardinia is known as a major centre of diversification of this species. As other legumes, this clover produces biologically active flavonoids including the subclass of isoflavones that are natural phytoestrogens with positive health effects. Present sources of isoflavones for medical/nutraceutical treatments are red clover (Trifolium pratense) and soybean (Glycine max). This study assessed the content and composition of flavonoids in 14 subterranean clover genotypes from Sardinia, grown ex‐situ in comparison with two red clover ecotypes, to acquire information on the potential of the species as an alternative source of isoflavones for possible exploitation. Twenty compounds were tentatively identified across the two clovers after HPLC and LC/ESI‐MS analyses, including clovamide, four flavonols, and 15 isoflavones. Most compounds were present as glucosides or glucosyl malonates. Subterranean clover extracts mainly comprised of derivatives of the isoflavones genistein, biochanin A, and formononetin. Compared to red clover, subterranean clover had higher content of total isoflavones and lower concentration of total flavonols. The isoflavone concentration in subterranean clover was higher than literature data for soybean or red clover. The existing genotypic variation warrants the possibility of selecting varieties with high isoflavone concentration for nutraceutical or pharmaceutical purposes.     

外源茉莉酸处理地下部对玉米化学防御反应影响的时间和浓度效应

在“高油115”玉米幼苗地下部施用4种不同浓度（10、50、100、200 μmol·L^-1）的茉莉酸3～48 h后,采用生化成分分析和基因表达分析方法,检测了处理部位（根系）和非处理部位（叶片）中防御物质及防御相关基因的表达,以探讨茉莉酸诱导玉米地下部对化学防御反应影响的时间和浓度效应.结果表明：茉莉酸处理玉米地下部对处理部位（根系）和非处理部位（叶片）化学防御反应的影响均与茉莉酸作用的时间和浓度相关.茉莉酸处理玉米地下部后,在3～12 h内就能直接诱导处理部位（根系）Bx9、PAL、PR-2a、MPI和FPS基因的表达,使得根中的丁布含量增加而总酚含量下降,其中100 μmol·L^-1茉莉酸处理浓度产生的诱导作用最强,其次是50 μmol·L^-1,再次是10 μmol·L^-1;后期诱导作用则逐渐减弱.另外,茉莉酸处理玉米地下部还能间接影响到非处理部位（叶片）的化学防御反应,50 μmol·L^-1茉莉酸在处理后3 h就能系统诱导叶片中Bx9和FPS基因的表达,使叶片的丁布含量增加;在6～24 h内则使叶片Bx9、PAL、PR-1、MPI和TPS基因表达量增加,丁布和总酚含量降低.可见,对于大部分检测指标来说,茉莉酸处理玉米地下部对地上部的间接诱导作用不如对地下部的直接诱导作用强,根系启动防御反应的时间较叶片早,同时,随着处理浓度的增加,茉莉酸对根系和叶片防御反应的诱导作用有增强趋势.     

Coordinate expression of AOS genes and JA accumulation: JA is not required for initiation of closing layer in wound healing tubers

Wounding induces a series of coordinated physiological responses essential for protection and healing of the damaged tissue. Wound-induced formation of jasmonic acid (JA) is important in defense responses in leaves, but comparatively little is known about the induction of JA biosynthesis and its role(s) in tuber wound-healing. In this study, the effects of wounding on JA content, expression of JA biosynthetic genes, and the involvement of JA in the initiation of closing layer formation in potato tubers were determined. In addition, the role of abscisic acid (ABA) in wound-induced JA accumulation was examined. The basal JA content in non-wounded tuber tissues was low (<3 ng g⁻¹ FW). Two hours after wounding, the JA content increased by >5-fold, reached a maximum between 4 and 6 h after wounding, and declined to near-basal levels thereafter. Tuber age (storage duration) had little effect on the pattern of JA accumulation. The expressions of the JA biosynthetic genes (StAOS2, StAOC, and StOPR3) were greatly increased by wounding reaching a maximum 2-4 h after wounding and declining thereafter. A 1-h aqueous wash of tuber discs immediately after wounding resulted in a 94% inhibition of wound-induced JA accumulation. Neither JA treatment nor inhibition of JA accumulation affected suberin polyphenolic accumulation during closing layer development indicating that JA was not essential for the initiation of primary suberization. ABA treatment did not restore JA accumulation in washed tuber tissues suggesting that leaching of endogenous ABA was either not involved or not solely involved in this loss of JA accumulation by washing. Collectively, these results indicate that JA is not required for the induction of processes essential to the initiation of suberization during closing layer development, but do not exclude the possibility that JA may be involved in other wound related responses.     

Proteolysis and cell death in clover leaves is induced by grazing

Summary.  Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 °C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 °C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms. Received June 24, 2002; accepted August 15, 2002; published online March 11, 2003     

叶片涂施茉莉酸对玉米幼苗防御反应的时间和浓度效应

茉莉酸是环境胁迫下植物产生防御反应的重要信号物质, 但它发挥生理作用的时间和浓度效应以及该效应在叶片和根系中差异性并不清楚。该文以‘高油115’玉米(Zea mays)为材料, 采用4种浓度(1、2.5、5和10 mmol·L^-1)的外源茉莉酸溶液涂施玉米幼苗叶片, 在3~48 h的不同时间内跟踪测定叶片和根系中的直接防御物质(丁布(DIMBOA)和总酚)含量及其合成调控基因(Bx1、Bx9和PAL)、直接防御蛋白调控基因(PR-1、PR-2a和MPI)和间接防御物质挥发物调控基因(FPS和TPS)表达的动态变化。结果表明, 外源茉莉酸处理对玉米叶和根系的化学防御反应具有显著的时间和浓度效应。茉莉酸处理玉米叶片后3~6 h就能诱导叶片中Bx9和PAL基因的表达, 使得丁布和总酚的含量显著增加, 且与处理浓度有呈正比的趋势, 随后诱导作用逐渐减弱; 茉莉酸处理还能明显诱导叶片中PR-2a和MPI基因的表达, 诱导作用分别持续到24和48 h; 在处理后3~6 h内, 高浓度茉莉酸处理对挥发物调控基因FPS表达起诱导作用, 而低浓度茉莉酸则对TPS基因的表达起诱导作用。此外, 茉莉酸处理玉米叶片还能间接影响到根系的防御反应, 但大部分检测指标表明间接诱导作用主要出现在处理后期(24~48 h)。例如, 在处理后48 h, 茉莉酸能系统增加根系中直接防御物质丁布和总酚的含量, 增强根系中防御相关基因PR-2a、MPI、FPS和TPS的表达, 并有随茉莉酸处理浓度的增加而增强的趋势。可见, 外源茉莉酸叶片涂施玉米幼苗对根系的间接诱导作用不如对叶片的直接诱导作用强; 叶片启动防御反应的时间较根系早; 随着处理浓度的增加, 茉莉酸对叶片和根系中防御反应的诱导作用有增强的趋势。     

蓟马取食、机械损伤以及外源水杨酸甲酯和茉莉酸对菜豆叶片防御酶活性的影响

【目的】探讨菜豆对昆虫取食防御反应的生化机制。【方法】研究了西花蓟马Frankliniella occidentalis取食、机械损伤以及外源水杨酸甲酯（MeSA）和茉莉酸（JA）处理后菜豆叶片防御酶活性的变化。【结果】西花蓟马取食、机械损伤及MeSA和JA处理均能明显提高过氧化物酶（POD）的活性,前2种处理POD活性在72 h上升到最高峰,而后2种处理则在48 h达到最高峰。蛋白酶抑制剂（PI）活性在西花蓟马取食后升高最明显。JA途径关键酶脂氧合酶（LOX）和多酚氧化酶（PPO）的活性在西花蓟马取食、机械损伤和JA诱导处理均升高,但外源MeSA诱导处理则不能诱导它们的活性(P>0.05)。SA途径的关键酶苯丙氨酸解氨酶（PAL）在西花蓟马取食和机械损伤后均有一个先升高后下降的过程,外源MeSA诱导只在24 h引起PAL活性升高,其余时间下和对照没有明显的区别,外源JA诱导未能引起PAL活性的显著变化(P>0.05)。西花蓟马取食、JA和MeSA诱导以及机械损伤均能诱导β-1,3 葡聚糖酶（PR-2）活性上升(P<0.05)。【结论】结果说明,不同处理可诱导菜豆植株产生明显的防御反应,但酶活性的变化与处理方式和处理时间有关。     

A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

Dynamics of the enhanced emissions of monoterpenes and methyl salicylate, and decreased uptake of formaldehyde, by Quercus ilex leaves after application of jasmonic acid

Jasmonic acid (JA) is a signalling compound with a key role in both stress and development in plants, and is reported to elicit the emission of volatile organic compounds (VOCs). Here we studied the dynamics of such emissions and the linkage with photosynthetic rates and stomatal conductance. We sprayed JA on leaves of the Mediterranean tree species Quercus ilex and measured the photosynthetic rates, stomatal conductances, and emissions and uptake of VOCs using proton transfer reaction mass spectrometry and gas chromatography after a dark-light transition. Jasmonic acid treatment delayed the induction of photosynthesis and stomatal conductance by approx. 20 min, and decreased them 24 h after spraying. Indications were found of both stomatal and nonstomatal limitations of photosynthesis. Monoterpene emissions were enhanced (20-30%) after JA spraying. Jasmonic acid also increased methyl salicylate (MeSa) emissions (more than twofold) 1 h after treatment, although after 24 h this effect had disappeared. Formaldehyde foliar uptake decreased significantly 24 h after JA treatment. Both biotic and abiotic stresses can thus affect plant VOC emissions through their strong impact on JA levels. Jasmonic acid-mediated increases in monoterpene and MeSa emissions might have a protective role when confronting biotic and abiotic stresses.     

白三叶和红三叶对人工融冻胁迫的生理响应差异

采用人工模拟融冻胁迫方法,通过测定白三叶(Trifolium repens)和红三叶(T.prat-ense)在融冻胁迫中叶片细胞膜透性、MDA含量、抗氧化酶(SOD、POD、CAT)活力、渗透调节物(脯氨酸、可溶性糖和蛋白质)含量变化,以揭示未来气候变化对三叶草的影响。结果表明,经历融冻胁迫循环后抗冻力强的白三叶植株能恢复生长,而抗冻力弱的红三叶枯萎死亡。在融冻阶段,两三叶草叶片细胞膜透性增大、抗氧化酶活力增高、MDA和渗透调节物含量大幅增加;在冻融阶段,两三叶草叶片细胞膜透性降低、MDA含量下降、抗氧化酶活力降低。但在融冻胁迫循环中,白三叶叶片POD和CAT活力高于红三叶,脯氨酸含量较红三叶高5倍,但细胞膜透性低于红三叶。白三叶在-5℃抗逆生理指标达到最大值,而红三叶在-10℃。白三叶对环境温度变化反应敏感,在-5℃通过快速激活抗氧化酶系统和积累渗透调节物以抑制膜脂过氧化和维护细胞水分平衡在融冻适应上起重要作用。白三叶具有较强的抗融冻能力,是未来值得应用推广的优良园林绿化植物。     

Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.     

Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.     

Transmitter-Like Release of Endogenous 3,4-Dihydroxyphenylalanine from Rat Striatal Slices

Biphasic electrical field stimulation (0.5-5 Hz, 2 ms, 25 V, 3 min) and high K+ (10-30 mM, 5 min) released endogenous 3,4-dihydroxyphenylalanine (DOPA) from superfused rat striatal slices. Characteristics of the DOPA release were compared with those of 3,4-dihydroxyphenylethylamine (dopamine, DA). Electrical stimulation at 2 Hz evoked DOPA and DA over similar time courses. alpha-Methyl-p-tyrosine (0.2 mM) markedly reduced release of DOPA but not of DA. Maximal release (0.3 pmol) of DOPA was obtained at 2 Hz and at 15 mM K+. The impulse-evoked release of DOPA and DA was completely tetrodotoxin (0.3 microM) sensitive and Ca2+ dependent and the 15 mM K+-evoked release was also Ca2+ dependent. On L-[3,5-3H]tyrosine (1 microM) superfusion, high K+ (15 and 60 mM) released DOPA and DA together with concentration-dependent decreases in tyrosine 3-monooxygenase (EC 1.14.16.2) activity as indicated by [3H]H2O formation, followed by concentration-dependent increases after DOPA and DA release ended. These findings suggest that striatal DOPA is released by a Ca2+-dependent excitation-secretion coupling process similar to that involved in transmitter release.     

Induction of HDMBOA-Glc accumulation and DIMBOA-Glc 4-O-methyltransferase by jasmonic acid in poaceous plants

Induction of the accumulation of 2-(2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (HDMBOA-Glc) by jasmonic acid (JA) was investigated in wheat, Job's tears (Coix lacryma-jobi), and rye. An increase in HDMBOA-Glc and a corresponding decrease in 2-(2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one)-beta-D-glucopyranose (DIMBOA-Glc) were found in wheat and Job's tears, whereas no such changes were observed in rye. The activity of S-adenosyl-L-methionine:DIMBOA-Glc 4-O-methyltransferase which catalyzes the conversion of DIMBOA-Glc to HDMBOA-Glc was detected in wheat leaves treated with 50 micro M JA. The activity started to increase 3 h after treatment with JA, reached a maximum after 9 h, and then decreased gradually. This mode of induction was well correlated with that for the accumulation of HDMBOA-Glc, indicating the induction of enzyme activity was responsible for the accumulation of HDMBOA-Glc. The enzyme was purified from JA-treated wheat leaves by three steps of chromatography, resulting in 95-fold purification. The enzyme showed strict substrate specificity for DIMBOA-Glc with a K(m) value of 0.12 mM. DIBOA-Glc was also accepted as substrate, but the K(m) value was 10 times larger than that for DIMBOA-Glc. The aglycones, DIMBOA and DIBOA, were not methylated by the enzyme. The K(m) value for S-adenosyl-L-methionine was 0.06 mM. The optimum pH and temperature were 7.5 and 35 degrees C, respectively. The activity was slightly enhanced by the presence of 1 mM EDTA, while heavy metal ions at 5 mM completely inhibited the activity.     

Induction of different isozymes of cytochrome P-450 and of microsomal epoxide hydrolase in rat liver by 2-acetylaminofluorene and structurally related compounds

The amounts of five different forms of cytochrome P-450 and of microsomal epoxide hydrolase were determined immunochemically in rat liver microsomes before and after treatment of the animals with 2-acetylaminofluorene and 15 structurally related compounds. The amount of cytochrome P-450c was found to be increased about 60-fold after treatment with 2-aminofluorene and 3-aminofluorene. Administration of 1-aminofluorene, 4-aminofluorene, 2-acetylaminofluorene and nitrofluorene increased this isozyme about 15-19 times. 2-Aminofluorene was found to inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a cytoplasmic receptor 50% at a concentration of 3.12 microM, while no such inhibition could be detected with 2-acetylaminofluorene. Induction of ethoxyresorufin O-deethylase activity was found to be highly correlated (+0.95) with the induction of cytochrome P-450c. Also correlated with the induction of this form was the amount of cytochrome P-450d (+0.84), which could be maximally increased about fourfold. Cytochromes P-450b + e were induced by 2-acetylaminofluorene, 4-acetylaminofluorene and fluorene (about tenfold), while 4-aminofluorene and 4-acetylaminofluorene were found to elevate cytochrome P-450PB/PCN-E about threefold. Microsomal epoxide hydrolase was induced by many of the compounds tested, with 2,7-diaminofluorene, 2,7-diacetylaminofluorene, 2-acetylaminofluorene and 2-(N-hydroxy)acetylaminofluorene being the most potent. No correlation of the induction of this enzyme with the induction of any isozyme of cytochrome P-450 was observed.     

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.     

Phyto-oestrogens in herbage and milk from cows grazing white clover, red clover, lucerne or chicory-rich pastures

A grazing experiment was carried out to study the concentration of phyto-oestrogens in herbage for cattle and in milk during two periods (May and June). Forty-eight Danish Holstein cows were divided into four groups with four treatment diets; white clover, red clover, lucerne and chicory-rich pastures. Each experimental period lasted 15 days. Herbage samples from the first day and individual milk samples from the last day of the experimental period were analysed for phyto-oestrogens using LC-MS technique. The total concentration of phyto-oestrogens was 21 399 mg/kg dry matter (DM) for red clover and 238 to 466 mg/kg DM for the other three herbages mainly due to a much higher concentration of biochanin A, formononetin and glycitein in red clover. In the milk, the total concentration of phyto-oestrogens was 253 to 397 μg/l for red clover milk and 56 to 91 μg/l in the milk from the other three treatments. This was especially due to a higher concentration of equol, daidzein and formononetin in the red clover milk. The concentration of biochanin A was significantly higher in milk from the red clover treatment in May while no differences were observed in June. Enterodiol was similar across treatments while the concentration of enterolactone was significantly lower for red clover milk compared with the other treatments. Of the tested pastures, red clover appears to have the highest concentration and to be the best source of phyto-oestrogens, especially equol, in bovine milk.     

Jasmonic acid elicitation of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones

Hypericum perforatum L. cell suspensions were evaluated for their viability, growth, dark gland formation and ability to produce phenylpropanoids and naphtodiantrones after elicitation with different jasmonic acid (JA) concentrations. Phenolic compounds were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization mass spectrometry (ESI-MS). The activities of two key enzymes of the phenylpropanoid/flavonoid pathways, phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) were also monitored to estimate general channeling in the different metabolic pathways. A 6-fold increase of phenolic compounds, flavanols and flavonols after JA elicitation was observed in cells. In contrast, anthocyanins were in lower amounts in JA treated cells suggesting a modification of the channeling in the phenylpropanoid pathway. Similar accumulations with maxima after 4 days of elicitation were found for naphtodianthrones (2.4-fold) such as hypericin and pseudohypericin in cells. At least a 6–8-fold increase of PAL and CHI activities was observed in JA elicited cells confirming a strong activation of the phenylpropanoid pathway. JA elicitation increased production of phenylpropanoids and naphtodianthrones in H. perforatum cell suspension without differentiation of dark glands under 16 h photoperiod.