Correlation between Phenylalanine Ammonia Lyase Activity and Phenolic Biosynthesis in p-Fluorophenylalanine-sensitive and -resistant Tobacco and Carrot Tissue Cultures

Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.     

Ultrasound-induced stress responses of Panax ginseng cells: enzymatic browning and phenolics production

The stress metabolic activities of Panax ginseng (P. ginseng) cells induced by low-energy ultrasound (US) were examined. P. ginseng cells in suspension cultures were exposed to 38.5 kHz US at two power levels (power density 13.7 and 61 mW/cm(3)) for 2 min. The US treatment caused rapid increase in the intracellular levels of polyphenol oxidase (PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) and the production of polyphenols (PP) and phenolic compounds. The US-induced enzyme activities and phenolics production are part of plant stress responses to a mechanical stimulus. The much higher PPO activity and rate of PP production in the sonicated cultures are correlated to enzymatic browning, suggestive of physical damage and membrane permeabilization of the cells by US. The cells after sonication also showed decreased water content and cell volume, which may also be attributed to US-induced cell membrane permeabilization and water release. High-pressure shock and fluid shear stress arising from acoustic cavitation were regarded as the major causes of the responses. Nevertheless, the US exposure caused only temporary cell growth depression but no net loss of biomass yield of the culture.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.     

Positive effects of temperature and growth conditions on enzymatic and antioxidant status in lettuce plants

The contents of two bioactive compounds (polyphenols and flavonoids) and their antioxidant and enzyme activities were determined in the leaves of six lettuce (Latuca sativa L.) cultivars subjected to 4 different day/night temperatures for 6 weeks.The total polyphenol and anthocyanin contents and the corresponding antioxidant activities were the highest at 13/10 °C and 20/13 °C, followed by 25/20 °C and 30/25 °C. The enzymatic activities of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were also the highest at low day/night temperatures, but the peroxidase (POD) activity was decreased at low day/night temperatures and increased at high day/night temperatures.The most significant positive correlation existed between anthocyanin content and PPO activity, total polyphenols and their antioxidant activities. The results showed that at relatively low temperatures, lettuce plants have a high antioxidant and enzymatic status. These results provide additional information for the lettuce growers.     

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.     

Peroxidase and phenylalanine ammonia-lyase activities,phenolic acid contents,and allelochemicals-inhibited root growth of soybean

The influence of the allelochemicals ferulic (FA) and vanillic (VA) acids on peroxidase (POD, EC 1.11.1.7) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activities and their relationships with phenolic acid (PhAs) contents and root growth of soybean (Glycine max (L.) Merr.) were examined. Three-day-old seedlings were cultivated in nutrient solution containing FA or VA (0.1 to 1 mM) for 48 h. Both compounds (at 0.5 and 1 mM) decreased root length (RL), fresh weight (FW) and dry weight (DW) and increased PhAs contents. At 0.5 and 1 mM, FA increased soluble POD activity (18% and 47%, respectively) and cell wall (CW)-bound POD activity (61% and 34%), while VA increased soluble POD activity (33% and 17%) but did not affect CW-bound POD activity. At 1 mM, FA increased (82%) while VA reduced (32%) PAL activities. The results are discussed on the basis of the role of these compounds on phenylpropanoid metabolism and root growth and suggest that the effects caused on POD and PAL activities are some of the many mechanisms by which allelochemicals influence plant growth.     

Effect of chlorsulfuron on phenylpropanoid metabolism in sunflower seedlings

The effect of the herbicide chlorsulfuron on phenylpropanoid titer and metabolism and the role of endogenous ethylene in this response was examined in light-grown sunflower (Helianthus annuus L.) seedlings. Application of chlorsulfuron to the apex resulted in large increases in both total phenolic and hydroxycinnamic acid content in hypocotyls isolated from the treated seedlings. Both of these parameters were increased within 24 h of herbicide treatment, and both reached a maximum level 3–4 days post-treatment. An increase in ethylene evolution was found to proceed in parallel with the alterations of phenolic content. The extractable activities of phenylalanine ammonia lyase,trans-cinnamic-4-hydroxylase, and soluble peroxidase were increased by chlorsulfuron treatment. Chlorsulfuron had little effect on total phenolic content when administered directly to isolated hypocotyl segments. Exogenous ethylene had no effect on the endogenous titer of phenolic compounds. Root-fed cobalt chloride (5 × 10^?4 M) inhibited chlorsulfuron-induced ethylene production by 92% and also inhibited the accumulation of phenolic materials by 56%. Exogenous ethylene was unable to reverse the inhibition caused by cobalt chloride. It was concluded that chlorsulfuron-induced increases in phenolic compounds were not mediated solely by endogenous ethylene.     

Correlation between Chlorophyll and Chlorogenic Acid Content in Tobacco Leaves

Developmental stages of tobacco (Nicotiana tabacum L. cv. Burley 21) flower and capsule were correlated with tissue contents of polyphenols and activities of phenylalanine ammonialyase, polyphenoloxidase, and peroxidase. Chlorogenic acid, scopolin, and scopoletin were present in most tissues, whereas rutin and two dihydroxyphenolic glycosides concentrated primarily in the corolla and placenta, respectively. Ovules contained only chlorogenic acid. As development progressed, polyphenols accounted for nearly 15% of the dry weight in the green capsule of field-grown plants. Fertilization triggered a rapid increase of chlorogenic acid in the ovary. When l-phenylalanine-U-¹⁴C was fed to the detached green capsules and capsule parts, an incorporation of radioactivity into chlorogenic acid and dihydroxyphenolic glycosides occurred which suggested in situ synthesis of these compounds. This was subtantiated by a positive correlation between phenylalanine ammonia-lyase activity and polyphenol accumulation. High polyphenoloxidase activity was associated mainly with the ovary, whereas peroxidase activity was maximal during senescence of all tissues. Polyacrylamide gel slab electrophoresis revealed five cathodic bands and one diffuse zone with poly-phenoloxidase activity in flower extracts. Two anodic poly-phenoloxidase isozymes appeared only in the fertilized ovary. Among 17 peroxidase isozymes, six cathodic forms were present throughout floral development, and the anodic ones increased in number and activity at the later stages of capsule growth.     

Changes in Formation and Localization of Phenolic Compounds in the Tissues of European and Canadian Yew during Dedifferentiation <Emphasis Type="Italic">In Vitro</Emphasis>

Changes in formation and localizations of phenolic compounds, including flavans, were investigated in the tissues of European and Canadian yew (Taxus baccata L. and T. canadensis Marsh.) during dedifferentiation in vitro. Annual shoots of European yew had the highest capacity for synthesizing these compounds. During the summer growth period, the content of total soluble phenolic compounds and flavans in these shoots was 30–40% higher than in the winter. Cell dedifferentiation and growth in vitro was accompanied by enhanced synthesis of phenolic compounds, including flavans, the change in tissue localization of these compounds, and an increase in the number of cells containing phenolics. Significant accumulation of phenolic compounds in callus cells resulted in necroses following two subcultures in the European and Canadian yew cultures initiated from summer explants, and following seven subcultures of the European yew calli initiated from winter explants. These data allow us to suggest that a high level of phenolic compounds in yew calli could be the reason for their necrosis.     

Changes of phenolic metabolism and oxidative status in nitrogen-deficient Matricaria chamomilla plants

The effects of nitrogen deficiency on selected physiological attributes, phenylalanine ammonia-lyase (PAL, EC. 4.3.1.5) activity, phenolic contents, peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities, lipid peroxidation status and H₂O₂ accumulation were studied in N-deficient Matricaria chamomilla (L.) over 12 days. N deficiency enhanced root growth and inhibited shoot growth. Chlorophyll composition and F _v/F _m were not affected by N stress, but nitrogen and soluble proteins decreased in both the rosettes and the roots. PAL activity, expressed per mg protein, was enhanced in N-deficient rosettes and tended to decrease by the end of the experiment, while in the roots PAL activity was maintained. Total phenolic contents increased in both rosettes and roots. Peroxidase and catalase activities in N-deficient rosettes tended to decrease by the end of the experiment, while in the roots they increased on the 12th day of deficiency. Furthermore, lipid peroxidation status increased in N-deficient roots on the 12th day, indicating that antioxidative protection was insufficient to scavenge reactive oxygen species being generated. Surprisingly, H₂O₂ content was even lower in N-deficient roots by the end of the experiment, while in the leaves increased. This observation in correlation to lipid peroxidation and H₂O₂ degradation is discussed. The importance of PAL activity and phenolic metabolites in combination with antioxidative enzymes for plant protection against oxidative stress and the significance of PAL activity for the mobilization of N availability in N-deficient tissue are also discussed in view of existing information.     

Quantitative analysis of polyphenols and antioxidant activity in four Daphne L. species

The content of biologically active phenolic compounds (total polyphenols, tannins, flavonoids, and phenolic acids) were determined using spectrophotometry in four wild Croatian species of Daphne L. in the family Thymelaeaceae (Daphne alpina, D. cneorum, D. laureola, and D. mezereum). The concentration of total flavonoids (TF) was highest in the leaves of these Daphne species (0.12?C0.51% dry herb weight, DW) whereas the content of other phenolic compounds analyzed were highest in the roots, including total polyphenols (TP; 2.71?C19.03% DW), tannins (T; 1.14?C7.39% DW), and total phenolic acids (TPA; 0.12?C0.87% DW). D. alpina contained the highest amount of polyphenols, with the exception of flavonoids, where maximum concentrations were found in D. laureola. We also examined the antioxidant activity of leaf, stem, and root extracts. All extracts analyzed demonstrated high free radical scavenging activity with the highest concentration in the leaf extracts of D. alpina. Leaf extracts of D. cneorum showed the highest antioxidant activity in a ??-carotene bleaching assay.     

Phenylalanine-ammoniaque lyase et composes phenoliques au cours de la maturation de la cerise

L-Phenylalanine-ammonia lyase (PAL) activity and phenolic content were investigated during growth and maturation of sweet cherry fruit on the tree. Phenolic acid, flavonoid and tannin contents, were studied in relation to PAL activity over two years. Concomitant variations in PAL activity and in flavonoid or phenolic acid concentrations were found during growth, suggesting that PAL is the major element controlling the synthesis of phenolic compounds in sweet cherry. During ripening, the increase in PAL activity did not appear to be directly related to anthocyanin synthesis. The concentrations of neochlorogenic acid and narcissin measured over the same period were also related to PAL activity.     

Wound-induced ethylene production, phenolic metabolism and susceptibility to russet spotting in iceberg lettuce

Mechanical wounding by cuts or punctures caused a brief increase in ethylene production by iceberg lettuce ( Lactuca sativa L.) leaf tissue. Wounding increased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, which was a function of the degree of injury. Wound-induced PAL activity appeared after 4 h and reached maximum activity in about 24 h before slowly declining to normal levels in about a week. A signal for PAL induction was transmitted at about 0.5 cm h⁻¹ from the site of injury to cells up to 2.5 cm away. Treatment with 100 μ2-aminoethoxyvinylglycine prevented wound-induced ethylene production but did not affect induced PAL activity. Injury increased the concentration of several soluble phenolic compounds that were easily oxidized to brown substances by polyphenol oxidase (EC 1.10.3.2) isolated from lettuce tissue. Wounding also increased peroxidase (EC 1.11.1.7) activity and lignin content, with cell wall lignification localized in wounded and adjacent cells. Although wounding alone did not induce russet spotting, it did greatly increase susceptibility to ethylene-induced russet spot development. In the presence of 3 μ1⁻¹ ethylene, the russet spot score increased as the degree of injury increased.     

Herbicide effects on phenolic metabolism in maize (Zea mays L.)and soybean (Glycine max L.) seedling

The activities of phenylalanine ammonia lyase [PAL; EC 4.3.1.5 [EC] ]and chalcone isomerase [Cl; EC 5.5.1.5 [EC] ] as well as the contentsof anthocyanin and total soluble hydroxyphenolic compounds wereinvestigated in maize (Zea mays L.) and soybean (Glycine maxL.) seedlings 120 h after treatment with the field dose of fiveherbicides from different groups (trifluralin, fluometuron,atrazine, alachlor, and rimsulfuron) having varied modes ofaction. The fresh weight of both species was greatly decreasedby trifluralin followed by fluometuron and atrazine. The dryweight was, in general, only slightly decreased by all the herbicideswith the largest response with trifluralin. On the other hand,the activities of PAL and Cl were greatly enhanced in both speciesby alachlor and rimsulfuron, but decreased by trifluralin. Fluometuroninduced decreases in PAL activity of maize only and decreasedCl activity of maize and soybean seedlings. Moreover, hydroxyphenoliccompounds were increased in both species by alachlor and rimsulfuronand decreased by trifluralin and atrazine. Similarly, anthocyanincontent was increased in both seedlings by alachlor and rimsulfuron,but decreased by trifluralin and fluometuron, whereas atrazinedecreased the anthocyanin content in maize only. The presentresults indicate that stress is maintained by the differentherbicides and confirm the controlling action of PAL and Clon the production of anthocyanin and phenolic compounds duringthe induced state of stress. In addition, dry weight reductionappeared to coincide with the changes in the parameters of secondarymetabolism, suggesting a regulatory role of secondary metabolismon seedling growth. Key words: Herbicides, phenylalanine ammonia lyase, chalcone isomerase, anthocyanin, hydroxyphenolics     

Phenolic acids and peroxidase activity in alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment

Treatment with ethephon increased the concentration of exogenous ethylene in Medicago sativa L. embryogenic cell suspension cultures (consisting of single cells, small cellular clumps and globular somatic embryos) and induced changes in the metabolism of phenolic substances, activities of peroxidase (EC 1.11.1.7) and caused significant suppression of suspension culture growth. Treatment with the ethylene-releasing substance, ethephon, resulted in a several-fold increase in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity above the basal level and was accompanied by an elevated accumulation of phenolic acids (significant increase of methoxy-substituted acids). The majority of newly synthesised phenolic acids was incorporated into the fractions of glycosides and esters bound to the cell wall. Phenolic glycosides seemed to serve as a metabolic pool from which the phenolics were utilised during further culture. The increased activity of wall-bound ionic peroxidase after ethephon application correlated with the pronounced incorporation of ferulic acid in the cell walls. In contrast, the increased level of exogenous ethylene did not influence the growth of culture of more advanced embryos nor did it significantly alter phenylpropanoid metabolism.     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

Changes in the levels of peroxidase and phenylalanine ammonia-lyase inBrassica napus cultivars showing variable resistance toLeptosphaeria maculans

Peroxidase and phenylalanine ammonia-lyase activities (PAL) were determined in leaves of healthy and inoculatedBrassica napus cultivars, showing differential disease reaction towards a virulent and a weakly virulent strain ofLeptosphaeria maculans, the black leg pathogen. Both enzymes showed increased activities as the result of inoculation, PAL activity increasing as early as 12 h after inoculation. The most significant increase in both peroxidase and PAL activity was observed when the moderately resistant cultivar, Cresor, was challenged with the weakly virulent strain. Highest activity of the two enzymes was detected 2 d after inoculation. Very low peroxidase activity was detected in both strains ofL. maculans, while no PAL activity was detectable in either of the strains. Cytochemical tests revealed increased peroxidase activity following inoculation, mainly in the epidermal and guard cells.     

Response of Winter Wheat to Cold: Production of Phenolic Compounds and L-Phenylalanine Ammonia Lyase Activity

The formation of soluble and polymeric (lignin) phenolic compounds, activity of L-phenylalanine ammonia lyase (PAL, EC 4.3.1.5), and content of free L-phenylalanine during cold hardening of winter wheat plants (Triticum aestivum L.) were studied. Cold treatment increased accumulation of soluble phenolic compounds in leaves while not affecting the content of lignin. The opposite was observed in tillering nodes. The activity of PAL was lower than in control plants in both tissues, and the content of free L-phenylalanine in tissues increased.     

Enhancement of phenolic compound production in Spirulina platensis by two-step batch mode cultivation

Phenolic compounds regarded as important pharmaceuticals with various biological activities are found in low amounts in microalgae. The objective of this study was to increase the amount of phenolic compounds in Spirulina platensis by a two-step batch mode cultivation. The evaluation of the effect of the sudden shift from low light to high light on phenolic compound production, antioxidant activity, growth, and biomass composition of S. platensis was undertaken. The amount of phenolic compounds was significantly increased by approximately eightfold (p?<?0.01) by the light treatment. There were also increases in total amounts of carbohydrate, phycocyanin, carotenoid, malondialdehyde, and antioxidant activities while there were significant decreases in total protein amounts (p?<?0.05). The relationships between antioxidant activities and total amounts of phenolic compounds were significantly correlated at the 99% confidence level (p?<?0.01) indicating that phenolic compounds were major contributors of antioxidant activities.