Phenolic acids associated with the resistance of lettuce cultivars to the lettuce root aphid

Phenolic acids, particularly caffeoylquinic acids, in lettuce root extracts were analysed by high pressure liquid chromatography (hplc), gas liquid chromatography (glc) and ultra-violet (uv) absorbance to seek a relationship with the resistance of certain lettuce cultivars to lettuce root aphid, Pemphigus bursarius. Although consistent results were obtained by each method, glc estimates of isochlorogenic acid tended to be low and uv estimates high. Hplc results were intermediate and since it was the easiest technique to perform routinely it was preferred. Isochlorogenic acid was the only caffeoylquinic acid detected in quantity and there were greater concentrations in resistant than in susceptible cultivars. Phenylalanine-ammonia lyase (PAL), the first enzyme in the phenyl propanoid pathway, is more active in resistant cultivars. Although these cultivars also had a greater tendency to browning when damaged, it was not due to greater polyphenol oxidase activity but probably to the presence of more isochlorogenic acid substrate. The results were consistent with an association between isochlorogenic acid concentration, PAL activity and resistance to P. bursarius.     

Induction of phenylalanine ammonia-lyase and increase in phenolics in lettuce leaves in relation to the development of russet spotting caused by ethylene

Russet spotting (RS), consisting of numerous small brown spots on the midrib of head lettuce (Lactuca sativa), is a physiological disorder induced by exposure to ethylene. In leaves suffering RS, the increase in spotting was accompanied by a parallel increase in the amount of phenolic compounds. Of these, chlorogenic acid and isochlorogenic acid were identified. Ethylene induced high phenylalanine ammonia-lyase (PAL) activity and RS formation in the susceptible cultivar Salinas, but not in the resistant cultivar Calmar. In the absence of ethylene neither significant PAL induction nor RS occurred. No correlation was found between the increase in polyphenol oxidase or peroxidase and the development of RS. The increase in PAL activity, however, was closely correlated with the development of RS. The increase in PAL activity preceded the development of RS, and the extent of RS was directly related to the level of PAL. Three temperatures (0.5, 5.5, and 12.5 C) were compared on the basis of their influence on both RS and PAL induction. At the lowest temperature (0.5 C) neither PAL induction nor RS occurred to a significant extent. At the highest temperature (12.5 C) an initial rapid increase in PAL activity and an earlier development of spotting were observed, but subsequently there was a decrease in both PAL activity and the rate of development of RS. At the medium temperature (5.5 C) both PAL activity and RS increased progresively with time. The decline of PAL activity at a higher temperature might be attributed to inactivation of the enzyme. Thus, a temperature favorable for induction of PAL activity by ethylene was also favorable for RS. These observations indicate a close interrelationship between the induction of PAL activity and the development of RS in response to ethylene, and suggest a causal relationship between the two events. PAL serves as a useful biochemical marker for the RS reaction.     

Regulation of phenylalanine ammonia-lyase activity and growth in lettuce by light and gibberellic acid

Abstract The effects of light and gibberellic acid (GA3) on growth and phenylalanine ammonia-lyase (PAL) activity were studied in seedlings of lettuce (Lactuca sativa L.). Using an in vivo assay for PAL it was shown that wounding caused by excising hypocotyls results in an increase in PAL activity with time that can mask the effect of light on the activity of this enzyme. When hypocotyl sections were excised from light-treated seedlings immediately prior to the in vivo assay of PAL, light was shown to cause a marked increase in PAL activity. Experiments with an inhibitor of PAL activity, α-aminooxy-β-phenylpropionic acid (AOPP), confirmed that the volatile radioactive products measured in the in vivo assay resulted from the activity of PAL. Gibberellic acid suppresses the light-induced increase in PAL activity and there is an inverse relationship between GA₃-induced growth and the activity of PAL. Over a wide range of GA₃ concentrations, the activity of PAL is also inversely correlated with growth rate along the length of the hypocotyl section; the upper halves of sections elongate more rapidly and have lower levels of PAL than the lower halves. Despite the strong correlation between growth and PAL activity, experiments with AOPP and t-cinnamic acid show that it is unlikely that elongation is regulated directly by products of PAL activity.     

不同甜叶菊品种叶中绿原酸类成分的比较研究

该文以14个扦插培育的甜叶菊品种叶为材料,从8种不同型号的树脂中筛选出一种合适的大孔吸附树脂对甜叶菊叶中绿原酸类成分进行纯化前处理,采用HPLC法对不同甜叶菊品种叶中所含绿原酸类成分进行比较分析。结果表明:(1)在8种不同型号的树脂中,XAD~(-1)6对甜叶菊叶中绿原酸类成分吸附-解析性能最佳。(2)经优化,上样液浓度1.20 mg·mL~(-1)、样品溶液pH 3、解析液乙醇体积分数70%时XAD~(-1)6树脂对甜叶菊叶中绿原酸类成分具有较好的纯化效果。(3) HPLC检测分析表明,在14个品种中共检测出新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C六种绿原酸类成分,其中主要成分均为异绿原酸A、绿原酸、异绿原酸C,而在品种3、5、13、14中没有检测出异绿原酸B。(4) 14个品种中6个绿原酸类成分的含量分别为异绿原酸A 20.55~54.3 mg·g~(-1)、绿原酸17.96~32.93 mg·g~(-1)、异绿原酸C 4.15~19.49 mg·g~(-1)、新原酸0.61~4.61 mg·g~(-1)、隐绿原酸0.52~3.11 mg·g~(-1)、异绿原酸B 0.0~3.17 mg·g~(-1),6种绿原酸类成分总量为43.9~97.8 mg·g~(-1)。可见,不同品种甜叶菊叶中绿原酸类成分含量有明显差异,富含绿原酸类成分的甜叶菊品种可用于开发获取绿原酸类物质。     

HPLC同时测定大叶冬青叶中10种多酚成分的含量及抗氧化活性研究

建立HPLC同时测定大叶冬青叶中新绿原酸、绿原酸、隐绿原酸、咖啡酸、对羟基肉桂酸、芦丁、异槲皮苷、异绿原酸B、异绿原酸A和异绿原酸C 10种多酚类化合物的定量分析方法,研究不同产地及不同采收时期大叶冬青叶中10种多酚类化合物的含量差异及其抗氧化活性。采用HPLC测定大叶冬青叶50%甲醇提取液中10种多酚类成分的含量,并选用DPPH法对其抗氧化活性进行初步探索。结果表明,10种多酚类化合物分离效果较好,标准曲线在检测范围内具有良好的线性(r>0.999 5),平均加样回收率在96.58%～112.03%,RSD<4%(n=6)。大叶冬青叶50%甲醇提取液抗氧活性良好。本实验建立的方法快速、准确、重复性好,可用于同时测定大叶冬青叶中新绿原酸、绿原酸、隐绿原酸、咖啡酸、对羟基肉桂酸、芦丁、异槲皮苷、异绿原酸B、异绿原酸A、异绿原酸C 10种成分的含量;不同产地、不同采收时期大叶冬青叶中10种多酚类化合物含量存在一定差异;不同来源大叶冬青叶50%甲醇提取液抗氧化能力具有明显差异。     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

Evolution des Teneurs en Acides Chlorogénique et "Isochlorogénique' au Cours du Traitement de Vernalisation des Racines de Cichorium intybus

In a cold-requiring variety of endive (chicory, Cichorium intybus L. ev. Witloof) we could demonstrate a relationship between the variations in the content of chlorogenic acid during the cold treatment of the roots and their disposition to produce flowers in vitro. An increase in the level of chlorogenic acid in the roots preceded the development of their aptitude to form flower buds. In addition, the three main forms of “isochlorogenic’ acid are present in the roots of this cultivar: 3,5-dicaffeoyl-quinic acid, 3,4-dicaffeoyl-quinic acid, and traces of 4,5-dicaffeoyl-quinic acid. The variations in the levels of chlorogenic and “isochlorogenic’ acids indicate that part of the chlorogenic acid (3-caffeoyl-quinic acid) is converted to 3,5-dicaffeoyl-quinic acid.     

Ca2+ is Required by Clubroot Resistant Turnip Cells for Transient Increases in PAL Activity that Follow Inoculation with Plasmodiophora brassicae

Phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity in clubroot disease‐resistant turnip calli was transiently increased by 20 h after the inoculation with Plasmodiophora brassicae spores. The magnitude of the increase in PAL activity was four to six times higher than constitutive PAL activity. There was no transient increase in PAL activity in susceptible calli. Preincubation of calli in Ca²⁺‐free medium or the removal of Ca²⁺ from cell surfaces by ethylene glycol bis(2‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐chelation, completely inhibited induced PAL activity. The influx of exogenous Ca²⁺ into cells appears necessary for this pathogen induced PAL activity. Verapamil and the calmodulin inhibitor W7 almost completely inhibited induced PAL activity at 1 and 0.1 mm , respectively. Neomycin, ruthenium red and (1‐(6‐[(17β‐3‐Methoxyestra‐1,3,5‐(10)‐trien‐17‐yl)amino]hexyl)‐1H‐pyrrole‐2,5‐dione) did not inhibit induced PAL activity. Thus, verapamil and N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride‐sensitive Ca²⁺‐mediated signalling process appear necessary for P. brassicae induced PAL activity. As the protein synthesis inhibitor cycloheximide (CHX) blocked the induced increasing PAL activity, de novo synthesis of PAL appears to be required for turnip cell defence reactions against P. brassicae.     

The correlation of phenylalanine ammonia-lyase and cinnamic acid-hydroxylase activity changes in Jerusalem artichoke tuber tissues

Summary Phenylalanine ammonia-lyase (PAL) and cinnamic acid-hydroxylase (CAH) exhibit parallel activity changes in tissues of Jerusalem artichoke (Helianthus tuberosus L.) tubers during ageing and in vitro culture. The activity of both enzymes appears in the tissue only after slicing and can be stimulated by manganese. Inhibition of CAH, by the deprivation of the substrate O₂, leads to cinnamic acid accumulation and PAL inhibition, a process prevented by cycloheximide. It is propounded that the link between PAL and CAH activities is the result of changes of the cinnamic acid pool size produced by a change in the activity of CAH.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.     

Intensity of radicle fluorescence as related to the resistance of seedlings of lettuce to the lettuce root aphid and carrot to the carrot fly

A technique is described which indicates by u.v. fluorescence the concentration of caffeoylquinic, chlorogenic and isochlorogenic acids in roots of germinating lettuce and carrot. The surface fluorescence of radicles from lettuce root aphid (Pemphigus bursarius) resistant seedlings was more intense than for susceptible seedlings, attributable to the higher concentration of isochlorogenic acid. In contrast, radicles of carrot seedlings resistant to carrot fly (Psila rosae) were less fluorescent than susceptible seedlings, corresponding to the lower concentration of chlorogenic acid in resistant seedlings. The technique is non-destructive and has been developed to distinguish between cultivars or breeding lines potentially resistant to these insect pests.     

Fungal endophytes-induced abscisic acid is required for flavonoid accumulation in suspension cells of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Treatment of suspension cells of Ginkgo biloba with fungal endophytes resulted in accumulation of flavonoids, increased abscisic acid (ABA) production and activation of phenylalanine ammonia-lyase (PAL). Fluridone, an inhibitor of ABA biosynthesis, was effective in inhibiting fungal endophytes-induced ABA biosynthesis, increase of PAL activity and flavonoids accumulation. Moreover, exogenous application of ABA enhanced PAL activity and increased accumulation of flavonoids in G. biloba cells with or without fungal endophytes elicitor. These finding suggest a causal relationship between ABA release and both PAL activity and flavonoid accumulation under fungal endophytes treatment and that ABA is involved in fungal endophytes-induced flavonoids accumulation in this plant.     

Exogenous application of L-phenylalanine and ferulic acid enhance phenylalanine ammonia lyase activity and accumulation of phenolic acids in pea (Pisum sativum) to offer protection against Erysiphe pisi

Phenylalanine ammonia lyase (PAL) activity was measured using HPLC in pea leaves following exogenous application of L-phenylalanine and ferulic acid. Treatment with different concentrations (50, 100, 150 ppm) of L-phenylalanine caused increased activity of PAL activity in comparison to control. In pea leaves treated with 50 ppm L-phenylalanine, maximum PAL activity was observed after 72 h of treatment. Application of ferulic acid first reduced PAL activity at lower concentration (50 ppm) but it further increased at higher concentrations of the compound (100 and 150 ppm) in pea leaves compared to control. Minimum PAL activity was 0.19 nM cinnamic acid/min/g fresh wt after 24 h at 50 ppm and then increased with time. Treatment with both compounds significantly increased the accumulation of phenolic acids and salicylic acid and reduced conidial germination of Erysiphe pisi on pea leaves. They were equally effective at 100 and 150 ppm in reducing conidial germination. Conidial germination on L-phenylalanine-treated leaves was 26% after 24 h and that on ferulic acid treated leaves 34% compared to control (46%). Foliar application of different concentrations of L-phenylalanine increased the level of ferulic acid in the leaves of pea plants. Maximum enzyme activity in terms of the accumulation of cinnamic acid (79.3 and 83.5 μg/g fresh wt) was observed following the application of L-phenylalanine after 24 and 48 h respectively. At 50 ppm, cinnamic acid accumulation in pea leaves was 35.6 and 39.4 μg/g fresh wt and 74.3 and 86.5 μg/g fresh wt at 100 ppm.     

Identification by high-performance liquid chromatography of tyrosine ammonia-lyase activity in purified fractions of Phaseolus vulgaris phenylalanine ammonia-lyase

Activities of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were assessed at each stage of a three-step purification of PAL. Assays were performed by high-performance liquid chromatographic (HPLC) separation and ultraviolet detection of reaction products. Use of HPLC permitted assay of low activities of PAL and TAL for periods up to approximately four and two days, respectively. HPLC also facilitated the accurate quantitation of the product of the TAL reaction, trans-p-coumaric acid, which was observed to isomerize readily under experimental conditions. PAL and TAL were associated throughout the purification procedure, with TAL activity at 0.6–1.3% of PAL activity. It was concluded that, contrary to previous reports, TAL and PAL activities are mediated by the same enzyme, or else by chromatographically very similar enzymes.     

Production of 5-methoxypodophyllotoxin in cell suspension cultures of Linum flavum L.

Cell suspension cultures of Linum flavum L., routinely grown on a NAA-containing medium, accumulated low levels of the phenylpropanoid-derived lignan 5-methoxypodophyllotoxin (5-MPT), up to 0.004% on a dry weight basis. Feeding experiments with the precursor L-phenylalanine resulted in a 3–5-fold increase in 5-MPT levels, but caused the levels of PAL activity to fall. Treatment of the cultures with the elicitor Nigeran, either alone or in combination with phenylalanine, caused the 5-MPT production to cease, even though PAL activity was rapidly enhanced by these treatments. Transfer of the cultures to NAA-free medium resulted in a 40–50 fold higher level of 5-MPT accumulation, the PAL activity levels being lowered compared to the routinely grown cells. With these more differentiated cultures, phenylalanine feeding and elicitor treatment, both on its own and in combination with the precursor, had no effect on 5-MPT production, even though the PAL activity levels were higher than in the untreated cells. It can be concluded that in lignan-accumulating cultures of L. flavum, PAL activity is nearly always detectable and seems to show a reciprocal relationship with 5-MPT accumulation.Abbreviations 5-MPT 5-methoxypodophyllotoxin - PAL phenylalanine ammonia lyase (EC 4:3:1.5) - NAA naphthaleneacetic acid     

蔓三七叶中分离绿原酸和异绿原酸及其抗氧化活性研究

以蔓三七叶为原料,制备其中的绿原酸和异绿原酸A、B和C,并对它们进行结构鉴定;同时,采用DPPH法、水杨酸比色法、邻苯三酚自氧化法来对其进行体外抗氧化活性研究。结果表明,分离制备的绿原酸和异绿原酸A、B和C质量分数分别为96. 8%、98. 2%、97. 6%和98. 7%。在0. 5～10μg/mL范围内,绿原酸和异绿原酸A、B和C对DPPH和羟基自由基表现出了较好的清除作用,随浓度升高而逐渐增大;但它们对超氧自由基的清除作用时,浓度从5μg/mL升高到100μg/mL时,清除作用都未出现明显的剂量依赖性,各样品与阳性对照组(VC)相比较而言,其清除超氧自由基的作用明显较弱。本文揭示了蔓三七叶中的绿原酸和异绿原酸A、B和C具有不同的抗氧化效果。     

An evaluation of phenylpropanoid metabolism during cold-induced sphagnorubin synthesis in Sphagnum magellanicum BRID

L-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Sphagnum magellanicum BRID. is inhibited by t-cinnamic acid in vitro only at relatively high doses. In contrast, p-coumaric acid does not display an inhibitory effect in a comparable concentration range. Sphagnum acid, an endogenous cinnamic acid derivative of sphagna, strongly enhances PAL activity at certain concentrations. The involvement of the phenylpropanoid pathway in the biosynthesis of the main reddish-violet wall pigment of Sphagnum magellanicum (sphagnorubin) is studied at several metabolic levels. Extractable PAL activity rises in response to the stimulus of sphagnorubin synthesis (nightly application of low temperature). If the formation of sphagnorubin is blocked in vivo by the PAL-inhibitor L--aminooxy--phenylpropionic acid (AOPP), complementation of the mosses by p-coumaric acid is able to overcome partially the inhibition. The mechanism of PAL induction by nightly cold treatment is independent of soluble carbohydrates which concomitantly accumulate as a result of photosynthetic action. Suppression of the sugar formation by application of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) is contrasted with an enhancement of PAL activity above the level of the merely cold-treated plants. The fluctuations of the enzyme level are principally unaffected by a DCMU-treatment.Abbreviations L-AOPP L--aminooxy--phenylpropionic acid - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5.) - TAL L-tyrosine ammonia-lyase Dedicated to Professor H. Rudolph     

The Activity-Integrated Method for Quality Assessment of Reduning Injection by On-Line DPPH-CE-DAD

A sensitive on-line DPPH-CE-DAD method was developed and validated for both screening and determining the concentration of seven antioxidants of Reduning injection. The pH and concentrations of buffer solution, SDS, β-CD and organic modifier were studied for the detection of DPPH and seven antioxidants. By on-line mixing DPPH and sample solution, a DPPH-CE method for testing the antioxidant activity of the complex matrix was successfully established and used to screen the antioxidant components of Reduning injection. Then, antioxidant components including caffeic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid were quantified by the newly established CE–DAD method. Finally, the total antioxidant activity and the multiple active components were selected as markers to evaluate the quality of Reduning injection. The results demonstrated that the on-line DPPH-CE-DAD method was reagent-saving, rapid and feasible for on-line simultaneous determination of total pharmacological activity and contents of multi-components samples. It was also a powerful method for evaluating the quality control and mechanism of action of TCM injection.