Nitrogen deficiency induced changes of free amino acids and coumarin contents in the leaves of Matricaria chamomilla

Through o-hydroxycinnamic acids, the biosynthesis of coumarins is connected with aromatic amino acid metabolism and nitrogen uptake. Therefore the quantitative changes in levels of some free amino acids and coumarins (herniarin and its glucosidic precursors (Z) - and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids; umbelliferone) in the leaf rosettes of chamomile (Matricaria chamomilla L.) subjected to nitrogen deficiency were studied. Nitrogen content decreased in the leaf rosettes and in the roots of N-deficient plants during the course of the experiment, but these plants produced significantly higher root biomass. Among secondary metabolites, the sum of 2-β-D-glucopyranosyloxy-4-methoxycinnamic acids increased sharply, herniarin increased slowly and the content of umbelliferone was low in N-deficient plants. We have concluded that nitrogen deficiency is not an inducing factor for stress accumulation of herniarin and umbelliferone. A decrease in levels of all detected amino acids, besides histidine, was found. Within aromatic amino acids, tyrosine was the most abundant. The content of free phenylalanine was significantly lower in both, control and N-deficient plants when compared to the content of tyrosine. In this view, the increase of herniarin glucosidic precursors is apparently due to enhancing phenylalanine ammonia-lyase activity under nitrogen deficiency and nitrogen-free carbon skeletons are shunted in to the phenylpropanoid metabolism, including biosynthesis of (Z)-and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids.     

Restoration of secondary metabolism in birch seedlings relieved from PAL-inhibitor

Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.     

Fe2O3 nanoparticles induced biochemical responses and expression of genes involved in rosmarinic acid biosynthesis pathway in Moldavian balm under salinity stress

The effect of iron oxide nanoparticle (NP) at four concentrations (0, 30, 60 and 90 ppm) and salinity at three levels (0, 50 and 100 mM) were investigated on rosmarinic acid (RA) production in 5-week-old Moldavian balm (Dracocephalum moldavica L.) plants. Salinity and spraying iron oxide NPs significantly affected tyrosine (Tyr), phenylalanine (Phe) and proline (Pro) amino acids content, Phenylalanine Ammonia-Lyase (PAL), Tyrosine Aminotransferase (TAT) and Rosmarinic Acid Synthase (RAS) genes expression levels, RA content, Polyphenol Oxidase (PPO), PAL and Superoxide Dismutase (SOD) activities, malondialdehyde (MDA) content and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. PAL, TAT and RAS genes expression rate and content of RA were enhanced in Moldavian balm plants exposed by NaCl + NPs. The results of high performance liquid chromatography (HPLC) revealed that simultaneous application of 50 mM NaCl and 90 ppm NPs increases the RA content in leaf by 81.15% as compared to control plants. The Tyr and Phe contents decreased in Moldavian balm plants exposed to salt stress. Application of NPs had a positive effect on the content of these amino acids. Proline content increased under salinity stress and application of iron NPs induced a significant increase in the Pro content of leaf. The results revealed that PAL, PPO and SOD enzymes activities increased under salinity conditions. The highest activity of PPO and SOD was observed in 100 mM NaCl + 60 ppm NPs treatment. Simultaneous application of 100 mM NaCl + 90 ppm NPs increased the MDA content and DPPH radical scavenging activity compared to control plants. It can be concluded that the application of appropriate levels of NPs moderates the effect of salinity stress in D. moldavica L. and results in an increased amount of RA compared to control plants.     

Matricaria chamomilla is not a hyperaccumulator, but tolerant to cadmium stress

The influence of low (3 μM) and high (60 and 120 μM) cadmium (Cd) concentrations were studied on selected aspects of metabolism in 4-week-old chamomile (Matricaria chamomilla L.) plants. After 10 days’ exposure, dry mass accumulation and nitrogen content were not significantly altered under any of the levels of Cd. However, there was a significant decline in chlorophyll and water content in the leaves. Among coumarin-related compounds, herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased significantly at all the levels of Cd tested. Cd did not have any effect on umbelliferone, a stress metabolite of chamomile. Lipid peroxidation was also not affected by even 120 μM Cd. Cd accumulation was approximately seven- (60 μM Cd treatment) to eleven- (120 μM Cd treatment) fold higher in the roots than that in the leaves. At high concentrations, it stimulated potassium leakage from the roots, while at the lowest concentration it could stimulate potassium uptake. The results supported the hypothesis that metabolism was altered only slightly under high Cd stress, indicating that chamomile is tolerant to this metal. Preferential Cd accumulation in the roots indicated that chamomile could not be classified as a hyperaccumulator and, therefore, it is unsuitable for phytoremediation.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Early defence reactions in Norway spruce seedlings inoculated with the mycorrhizal fungus <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis> (Persoon) Coker &; Couch and the pathogen <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> (Fr.) Bref.

The activities of the enzymes responsible for cell-wall strengthening and salicylic acid (SA) content in Norway spruce seedlings were investigated after inoculation with the ectomycorrhizal fungus Pisolithus tinctorius or the pathogen Heterobasidion annosum, and after treatment with elicitors from both of these fungi. Inoculation with both fungi increased guaiacol peroxidase (POD) activity in the roots of the pathogen-inoculated seedlings during the earliest phases of colonisation, and induced the activities of several POD isoforms. Two of these were only seen in pathogen-inoculated seedlings and corresponded with increased POD activity against ferulic acid. Colonisation with H. annosum triggered an increase in phenylalanine ammonia lyase (PAL) activity in the roots of the spruce seedlings, which was followed by an accumulation of free SA. One month after inoculation levels of free SA were increased also in the shoots of H. annosum-inoculated seedlings. In contrast increase in free SA content in the roots of P. tinctorius-inoculated seedlings was only transient. Similarly to inoculation, treatment with elicitors of H. annosum increased the PAL and POD activity, as well as SA content in the roots of spruce seedlings. A positive correlation between PAL activity and SA content in the H. annosum-inoculated seedlings and accumulation of SA precursors in the phenylpropanoid pathway indicate that the plant defence mechanisms, during which SA is synthesised through the PAL pathway, are exploited by H. annosum for facilitation of colonisation.     

Thin layer chromatographical detection of tyrosine produced from <Emphasis Type="SmallCaps">l</Emphasis>-[U-<Superscript>14</Superscript>C]phenylalanine by ruminal microbes

Summary.  Thin layer chromatographical detection of tyrosine (Tyr) synthesized from l-[U-¹⁴C]phenylalanine (Phe) (1 mM) by rumen bacteria (B) and protozoa (P) collected from fistulated Japanese Goat was carried out. About 16 and 12% of the added Phe was converted to Tyr by B and P, respectively. Large amount of radioactivity in ether fractions indicated an abundant production of aromatic acids from Phe. Small amount of radioactivity found in CO₂ fractions implied an occurrence of considerable decarboxylation reaction(s) by rumen bacteria and protozoa. Received July 18, 2001 Accepted December 3, 2001     

High nitrogen supply affects the metabolism of Matricaria chamomilla leaves

N-status of the two Matricaria chamomilla cultivars grown in the presence of high potassium nitrate concentration was evaluated and compared with ammonium nitrate supply. After 5 days of potassium nitrate treatment the visible increase of dry mass together with total chlorophyll accumulation were observed. In both cultivars, ammonium nitrate application led to increased accumulation of N-containing compounds in chamomile leaves. NH₄NO₃ nitrogen supply influenced activity of nitrate reductase positively. In vivo nitrate reductase activity reached maximum in lower nitrate supply and decreased in higher nitrate availability significantly. Among the most abundant leaf secondary metabolites, the high nitrate availability both KNO₃ and NH₄NO₃ significantly increased umbelliferone level. The highest potassium nitrate dose (60 mmol per plant) caused an osmotic stress accompanied with lower tissue water content and turgor loss. In such condition the decrease in (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acid, herniarin and dicycloethers, as well as PAL activity was observed. On the other hand, strong increase of umbelliferone is likely a stress response and is related to its antioxidant activity.     

The conversion of [3H] tryptophan to 5-[3H] hydroxytryptamine in mouse brain following depletion of phenylalanine and tyrosine.

Abstract— Phenylalanine ammmonia-lyase (PAL), an enzyme which converts phenylalanine (Phe) and tyrosine (Tyr) to trans-p-cinnamic acid and trans-p-coumaric acid, respectively, was administered to mice and its effect on the conversion of [³H]tryptophan to 5-[³H]HT in the brain was measured. Although PAL significantly depleted plasma Tyr, it has little or no effect on either brain Tyr or catecholamine concentrations. Endogenous brain tryptophan levels were significantly increased 2 h after PAL administration, brain 5-HT was dramatically increased 4 h following PAL and each returned to baseline levels by 8 h. This return to baseline was accompanied by a marked decrease in the fraction of tryptophan converted to 5-HT during a 20 min pulse period preceding death, suggesting the activation of a compensatory decrease in 5-HT synthesis in response to increased 5-HT concentration. These data suggest that PAL administration readily produces reversible alterations in 5-HT synthesis and that this may be a fruitful approach to studying brain 5-HT function.     

Salicylic acid-induced changes to growth and phenolic metabolism in <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> plants

The influence of salicylic acid (SA) doses of 50 and 250 μM, for a period of up to 7 days, on selected physiological aspects and the phenolic metabolism of Matricaria chamomilla plants was studied. SA exhibited both growth-promoting (50 μM) and growth-inhibiting (250 μM) properties, the latter being correlated with decrease of chlorophylls, water content and soluble proteins. In terms of phenolic metabolism, it seems that the higher SA dose has a toxic effect, based on the sharp increase in phenylalanine ammonia-lyase (PAL) activity (24 h after application), which is followed by an increase in total soluble phenolics, lignin accumulation and the majority of the 11 detected phenolic acids. Guaiacol-peroxidase activity was elevated throughout the experiment in 250 μM SA-treated plants. In turn, some responses can be explained by mechanisms associated with oxidative stress tolerance; these mitigate acute SA stress (which is indicated by an increase in malondialdehyde content). However, PAL activity decreased with prolonged exposure to SA, indicating its inhibition. Accumulation of coumarin-related compounds (umbelliferone and herniarin) was not affected by SA treatments, while (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids increased in the 250 μM SA-treated rosettes. Free SA content in the rosettes increased significantly only in the 250 μM SA treatment, with levels tending to decrease towards the end of the experiment and the opposite trend was observed in the roots.     

Circadian rhythm of (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxy cinnamic acids and herniarin in leaves of Matricaria chamomilla

Chamomile (Matricaria chamomilla) in the above-ground organs synthesizes and accumulates (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxy cinnamic acids (GMCA), the precursors of phytoanticipin herniarin (7-methoxycoumarin). The diurnal rhythmicity of the sum of GMCA (maximum before daybreak) and herniarin (acrophase at 10 h 21 min of circadian time) was observed under artificial lighting conditions LD 12:12. The acrophase is the time point of the maximum of the sinusoidal curve fitted to the experimental data. In continuous light, the circadian rhythms of both compounds were first described with similar acrophases of endogenous rhythms; a significantly different result from that in synchronized conditions. The rhythms’ mesor (the mean value of the sinusoidal curve fitted to the experimental data) under free-running conditions was not influenced. Abiotic stress under synchronized conditions decreased the average content of GMCA to half of the original level and eliminated the rhythmicity. In contrast, the rhythm of herniarin continued, though its content significantly increased. Nitrogen deficiency resulted in a significant increase in GMCA content, which did not manifest any rhythmicity while the rhythm of herniarin continued. Circadian control of herniarin could be considered as a component of the plant’s specialized defence mechanisms.     

Biotransformation of phenylpyruvic acid to phenyllactic acid by growing and resting cells of a <Emphasis Type="Italic">Lactobacillus</Emphasis> sp.

Phenyllactic acid (PLA) is a novel antimicrobial compound derived from phenylalanine (Phe). Lactobacillus sp. SK007, having high PLA-producing ability, was isolated from Chinese traditional pickles. When 6.1 mM phenylpyruvic acid (PPA) was used to replace Phe as substrate at the same concentration, PLA production increased 14-fold and the fermentation time decreased from 72 h to 24 h with growing cells. With resting cells, however, 6.8 mM PLA could be obtained as optimal yield using the following conditions: 12 mM PPA, 55 mM glucose, pH 7.5, 35°C and 4 h.     

Nitric oxide signals ROS scavenger-mediated enhancement of PAL activity in nitrogen-deficient Matricaria chamomilla roots: side effects of scavengers

Owing to the abundance of phenolic metabolites in plant tissue, their accumulation represents an important tool for stress protection. However, the regulation of phenolic metabolism is still poorly known. The regulatory role of reactive oxygen species (ROS) in the activity of phenylalanine ammonia-lyase (PAL) in nitrogen (N)-deficient chamomile roots treated for 24 h was studied using three ROS scavengers [dithiothreitol (DTT), salicylhydroxamic acid, and sodium benzoate]. Scavengers decreased the level of hydrogen peroxide and/or superoxide (and up-regulated ascorbate/guaiacol peroxidase and glutathione reductase), but, surprisingly, stimulated PAL activity. This up-regulation was correlated with increases in nitric oxide (NO) content, total soluble phenols, selected phenolic acids, and, partially, lignin (being expressed the most in DTT-exposed roots). We therefore tested the hypothesis that NO may be involved in these changes. Application of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) decreased PAL activity and the accumulation of soluble phenols in all treatments. Exogenous H₂O₂ and NO also stimulated PAL activity and the accumulation of phenols. We conclude that NO, in addition to hydrogen peroxide, may regulate PAL activity during N deficiency. The anomalous effect of PTIO on NO content and possible mechanism of ROS scavenger-evoked NO increases in light of the current knowledge are also discussed.     

The effect of phenylalanine on biosynthesis of protoberberine alkaloids in the cell culture of low meadow-rue

The addition of 7 mM phenylalanine to the nutrient medium for low meadow-rue (Thalictrum minus L.) cell culturing on the 7th or 8th day doubled berberine secretion into medium. Simultaneously, the content of phenolic compounds increased in the cells and medium. Investigation of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activities showed that exogenous Phe activated PAL by 35% and inactivated TAL by 20%. When the crude extract was separated on DEAE-Sephacel column, two proteins were isolated. One of them displayed both PAL and TAL activities, whereas another protein displayed only PAL activity. This activity disappeared after cell culturing longer than 20 days and also under the effect of Phe at a concentration reducing alkaloid biosynthesis. Phe addition to medium also increased the content of protein in both the cells and culture medium. The proportion of low-molecular proteins in the medium increased. Testing antimicrobial activity of the medium showed that it was determined by berberine and to a lesser degree by palmatine. Protein fraction also demonstrated antimicrobial activity. An improved antimicrobial activity after Phe adding to medium resulted from alkaloid and protein accumulation. The conclusion was made that one of the mechanisms of Phe action was the control of alkaloid biosynthesis with the involvement of the enzyme system of the early steps of the phenylpropanoid pathway, which, in its turn, is one of the stages in stress-induced plant response to pathogen action.     

Solvent selection and optimization of α‐chymotrypsin‐catalyzed synthesis of N‐Ac‐Phe‐Tyr‐NH2 using mixture design and response surface methodology

A peptide, N‐Ac‐Phe‐Tyr‐NH₂, with angiotensin I‐converting enzyme (ACE) inhibitor activity was synthesized by an α‐chymotrypsin‐catalyzed condensation reaction of N‐acetyl phenylalanine ethyl ester (N‐Ac‐Phe‐OEt) and tyrosinamide (Tyr‐NH₂). Three kinds of solvents: a Tris–HCl buffer (80 mM, pH 9.0), dimethylsulfoxide (DMSO), and acetonitrile were employed in this study. The optimum reaction solvent component was determined by simplex centroid mixture design. The synthesis efficiency was enhanced in an organic‐aqueous solvent (Tris‐HCl buffer: DMSO: acetonitrile = 2:1:1) in which 73.55% of the yield of N‐Ac‐Phe‐Tyr‐NH₂ could be achieved. Furthermore, the effect of reaction parameters on the yield was evaluated by response surface methodology (RSM) using a central composite rotatable design (CCRD). Based on a ridge max analysis, the optimum condition for this peptide synthesis included a reaction time of 7.4 min, a reaction temperature of 28.1°C, an enzyme activity of 98.9 U, and a substrate molar ratio (Phe:Tyr) of 1:2.8. The predicted and the actual (experimental) yields were 87.6 and 85.5%, respectively. The experimental design and RSM performed well in the optimization of synthesis of N‐Ac‐Phe‐Tyr‐NH₂, so it is expected to be an effective method for obtaining a good yield of enzymatic peptide. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Significance of phenols in cadmium and nickel uptake

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols.     

Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs) biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL).     

Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNAl (Osakabe et al., 1995, Plant Sci. 105: 217–226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover,the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.Abbreviations IgG immunoglobulin G - IPTG isopropylthio--d-galactoside - PAL phenylalanine ammonia-lyase The authors thank Dr. Kunio Hata of Nippon Paper Industries Co., Ltd. (Japan) for supplying P. kitakamiensis. This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (No. 07406008).     

The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue

During the first 24 h of in vitro incubation of excised potato tuber (Solanum tuberosum L.) discs, the appearance of phenylalanine ammonia-lyase (PAL; EC 3.4.1.5) and the accumulation of chlorogenic acid are both stimulated by infection with Phytophthora infestans (Mont.) de Bary. Whereas in control tissue the level of PAL reached a stable plateau value after 40 h, in infected tissue it subsequently rose again, in one experiment, as the fungal mycelium developed. In the infected but not the control tissue, the level of chlorogenic acid subsequently fell to about to about 20% of its maximum after 50 h. The time courses of increases in cinnamic acid 4-hydroxylase (CA4H; EC 1.14.13.11; 0–60 h) and of caffeic acid acid o-methyltransferase (COMT; EC 2.1.1.42; 0–160 h) are not altered by fungal infection. If the discs are restored to the tuber environment immediately after excision, by placing them inside a host tuber, the activity of PAL as well as those of CA4H and COMT remained at the constant low endogenous level for at least 60 h, irrespective of whether the discs had first been inoculated with P. infestans. The increase in PAL may not be an obligatory feature of the P. infestans/potato compatible interaction but dependent on an underlying wound response. The experiments provide further evidence that PAL is the rate limiting step of chlorogenic acid biosynthesis in potato tuber discs.Abbreviations PAL phenylalanine ammonia-lyase - CA4H cinnamic acid 4-hydroxylase - COMT caffeic acid o-methyltransferase - CGA chlrogenic acid (5-o-caffeoylquinic acid) - gfwt gram fresh weight     

Paclitaxel production is enhanced in suspension‐cultured hazel (Corylus avellana L.) cells by using a combination of sugar,precursor, and elicitor

Hazel (Corylus avellana L.) has recently been drawing attention as an alternative source of taxol. In the present study, the effects of sugar type, and different concentrations of phenylalanine (Phe) and vanadyl sulfate (V) on the production of taxol in C. avellana were investigated. A factorial experiment was used to optimize the concentrations of the precursor and elicitor. The cells were treated with Phe and V on the fourth day of culture and were harvested every 2 days until the 10th day. By increasing the Phe and V supply, taxol production increased during the culture period and the maximum level of 4.2 μg/g (dry weight) was obtained at day 10 by combining 3 μM of Phe and 0.05 and 0.1 mM of V in media supplemented with fructose (3%). The time course study on taxol production suggested that the appropriate time for using Phe is day 4 of culture, and day 8 for V. Overall, taxol production in C. avellana cell suspension culture was improved by the use of the combined strategy.