Role de la phenylalanine dans la biosynthese des flavonoides et acides “cinnamique” dans les tiges volubiles de Periploca Graeca

Phenylalanine is the precursor of the cinnamic acids and coumarins in the stems and leafs of P. graeca L. Esterification of p-coumaric acid by quinic acid is required before oxidation to chlorogenic acid. In our experiments, we did not obtain radioactive flavonols from ¹⁴C phenylalanine. PAL activity varies as a result of light and temperature in the same manner as the level of flavonoids (especially the phenolic acids). This enzyme, therefore, plays a regulatory role in the synthesis of these phenolic substances. The variation in PAL activity during illumination does not follow the same course as described for other plants.     

Sur la presence de deux isoenzymes de la phenylalanine-ammoniac-lyase chez Aesculus hippocastanum

By using the normal techniques of protein separation, two forms of PAL with different properties have been separated from the glandular tissue of Aesculus hippocastanum. A hypothesis is proposed for correlating the isoenzyme activities with the accumulation of flavonoid compounds and benzoic acids in the trichomes.     

Density-labelling studies on the photocontrol of phenylalanine ammonia lyase levels in Cucumis sativus

The de novo synthesis of PAL is demonstrated to occur sometime between imbibition and the end of a 4-hr white light treatment. H₂OD₂O transfer experiments indicate that PAL synthesis may occur during the light period whilst D₂O-H₂O transfer experiments indicate that synthesis of inactive PAL may occur during dark growth followed by activation by light. Neither of these observations is conclusive. De novo synthesis of PAL occurs in excised hypocotyls of gherkin and tuber discs of potato either in darkness or in light. It is concluded that there is as yet no evidence which definitively shows that light controls PAL levels by regulating the rate of de novo synthesis.     

深红酵母转化反式肉桂酸生成L-苯丙氨酸的研究

研究了深红酵母Ａｓ２．２７９产生Ｌ－苯丙氨酸解氨酸（ＰＡＬ）的条件、转化反式 桂酸（ｔＣａ）生成Ｌ－苯丙氨酸（Ｌ－Ｐｈｅ）的条件以及几种因素对ＰＡＬ稳定性的影响,结果表明,最佳转化条件为：１．０％ｔ－Ｃａ,８ｍｏｌ／Ｌ氨,ｐＨ１０．０,３０℃。在转化液中加入还原剂和充入Ｎ２有利于提高酶的稳定性,在此条件下可一次转化６４％的ｔ－Ｃａ,保留６０％的酶活。生成Ｌ－Ｐｈｅ浓度为５．８ｇ／Ｌ。     

Evidence for a pool of inactive phenylalanine ammonia-lyase in Cucumis sativus seedlings

Evidence is presented which suggests that an inactive form of PAL exists in dark grown gherkin seedlings and may be activated by the application of protein synthesis inhibitors. The significance of this finding is discussed with reference to current views on the regulation of PAL activity.     

Effect of ageing on enzymes of phenylpropanoid metabolism in Solanum tuberosum discs

-Separation of cell fractions or cell organelles of potato tuber by differential centrifugation and by sucrose density gradient centrifugation showed that, in dormant tissue, nearly all the activity of shikimate and prephenate dehydrogenases, phenylalanine ammonia lyase, cinnamate-4-hydroxylase and an O-methyltransferase for caffeate was in the soluble fraction. All these enzymes increased in activity in slices aged in light for 18 hr. In contrast to the other enzymes, cinnamate hydroxylase becomes associated with the microsomal fraction in aged discs.     

Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae

Suspension cultured cells of a blast-resistant rice genotype (Oryza sativa L. cv. Gigante Vercelli) were treated with cell wall hydrolysates prepared from the fungal pathogen Magnaporthe oryzae. As a consequence, a complex pattern of phenylalanine ammonia lyase time course specific activity levels was evident. Ion-exchange chromatographic fractionation of crude extracts suggested that the early (6 h) and the late (48-72 h after elicitation) increase of activity relied upon the sequential induction of two different isoenzymes. The relative expression levels of 11 genes putatively coding for a phenylalanine ammonia lyase were measured by semi-quantitative capillary gel electrophoresis of RT-PCR products. Two genes were indeed found to be induced by treatments with the hydrolysate, and data were validated by real-time PCR. Conversely, only the early-responsive enzyme form was observed following elicitation in a blast-sensitive rice genotype (cv. Vialone nano). Therefore, the late-responsive isoform may represent a candidate gene to select for decreased sensitivity to blast.     

Effects of glyphosate on phenolic metabolism in yellow nutsedge leaves

The action of the herbicide glyphosate [N-(phosphonomethyl)-glycine] on phenolic metabolism and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity was investigated in yellow nutsedge (Cyperus esculentus L.). Glyphosate caused significant increases in the amount of total soluble hydroxyphenolics in the three fractions studied (neutral, acid and residual). Qualitative and quantitative differences in relation to these fractions and the amount of applied glyphosate were observed. Most of the phenolic compounds which increased after glyphosate treatment were benzoic acids (gentisic. p -OH-benzoic, salicylic and vanillic). Gentisic acid showed the greatest increase in neutral and acid fractions, being twenty- and four-fold, respectively, of the amount found in the control. PAL activity was not affected by the lowest doses of glyphosate (10−⁴and 10−³ M) , but a dramatic decrease in PAL activity was observed after 10−² M treatment. These findings, together with the low levels of cinnamic acids measured in treated yellow nutsedge plants, suggest that PAL activity is only marginally involved in glyphosate action. Since the herbicidal action probably takes place at 5-enol-pyruvylshikimate-3-P synthase (EC 2.5.1.19), an alternative pathway to PAL in phenolic biosynthesis should be activated yielding benzoic acids.     

Mechanism of phytochrome action in the control of biosynthesis of anthocyanin in Brassica oleracea

-The synthesis of anthocyanin in red-cabbage is very sensitive to control by light, R/FR reversibility being effected by exposures of 5 min duration, The demonstration of this control does not depend upon a preceding irradiation of high-intensity light but depends upon the duration of incubation in darkness subsequent to irradiation. R/FR reversibility is well shown in seedlings kept in darkness for 48 hr after exposure but after 120 hr this reversibility is no longer evident. This is due to the fact that a further synthesis of anthocyanin occurs in unexposed seedlings and in FR and R/FR treated material in the period from 48 to 120 hr but does not occur in the R treatment after 48 hr. Reagents such as n-propanol which are believed to increase membrane permeability, greatly increase anthocyanin synthesis in dark grown material. n-PrOH also reverses the effect of 5 min FR irradiation but, by contrast with R light, does not promote PAL activity. It is concluded that the limitation to synthesis in material unexposed to light is substrate availability at the site of flavonoid biosynthesis, rather than the level of PAL activity. The evidence presented supports the hypothesis that R/FR reversible phytochrome action involves the control of the passage of substrate through a membrane to the site of anthocyanin biosynthesis.     

Organ- and Tissue-specific Biosynthesis of Flavonoids in Seedlings of Oenothera odorata (Onagraceae)

In developing Oenothera odorata seedlings, phytochrome-mediated accumulation of various flavonoids (mainly glycosides of cyanidin and quercetin) is detectable, subsequent to a transient induction of the key enzymes of the general phenylpropanoid metabolism, L-phenylalanine ammonia lyase (PAL) and of flavonoid biosynthesis, chalcone synthase (CHS). Organ- and tissue-specific distribution of these enzymes and of the flavonoid end products was investigated in seedlings, irradiated with continuous far-red light. Anthocyanins and quercetin glycosides are mainly localized in both the upper and lower epidermis of the cotyledons and to a lesser extent also in the epidermal cell layer of the hypocotyl. An obvious organ-specific distribution was observed for the anthocyanins: cyanidin-3,5-O-diglucoside accumulates in the epidermal cells of the cotyledons, whereas cyanidin-3-O-glucoside is restricted to the epidermis of the hypocotyl. By contrast the pattern of quercetin glycosides is the same in the cotyledons and in the hypocotyl. The methylated flavonol aglycone 3-0-methylquercetin was found to be localized in the seed coat. According to this organ- and tissue-specific pattern of flavonoids, immunochemical and immunohistochemical detection of PAL and CHS revealed a predominant localization of theenzymes in the epidermal layers of the cotyledons and the hypocotyl but also in the cells surrounding the vascular bundles. The role of compartmentation in regulation of flavonoid biosynthesis and putative functions of flavonoid compounds are discussed.     

Metabolism of aromatic acids by Polyporus hispidus

When grown on glucose as principal carbon source the culture medium of Polyporus hispidus was found to contain phenolic acids, including p-coumaric and caffeic acids. ¹⁴C-Studies indicated that phenylalanine is converted to cinnamic acid as well as to phenylpyruvic acid and tyrosine in cultures. Cell-free preparations of mycelium contained phenylalanine and tyrosine ammonia-lyse activities and were capable of effecting the hydroxylation of cinnamic, p-coumaric and benzoic acids.     

Major resin acids of Pinus nigra needles

Labdane diterpene acids were found to be the major resin acid components in Pinus nigra needles of various seed sources. The major constituents have been identified as 4-epiimbricataloic acid, manoyl oxide 19-oic acid, 4-epicommunic acid, and 15-monomethyl pinifolate. A GC method was developed to analytically differentiate pinifolic acid from its monomethyl ester in an admixture of both compounds. A minor resin acid was identified as 18-acetoxy-8(17)-labden-15-oic acid. 10-Nonacosanol and isoabienol were identified as major constituents of the needle and cortex extractives, respectively.     

Control at the level of substrate supply—an alternative in the regulation of phenylpropanoid accumulation in plant cells

The problem of the intracellular mechanisms responsible for the control of accumulation of phenylalanine-derived polyphenols in plants is considered. Possible control functions of phenylalanine ammonia lyase (PAL) in this process are critically discussed and experimental facts are surveyed providing evidence that substrate (phenylalanine) supply rather than enzymic (PAL) activity is the most likely limiting factor in controlling phenylpropanoid accumulation. These facts involve: frequent lack of consistent correlation between changes in the level of PAL and accumulation rate of phenylpropanoids, high deaminating capacity of PAL markedly exceeding the level required to sustain the formation of polyphenols in most tissues, stimulatory action of phenylalanine on polyphenol accumulation when administered externally, very low and constant levels of free endogenous phenylalanine in plant tissues, and the existence of balanced relationship between protein metabolism and the formation of flavonoids and cinnamic acid derivatives in plants.     

Regulation of polyphenols accumulation by combined overexpression/silencing key enzymes of phyenylpropanoid pathway

There is a growing interest in the metabolic engineering of plant with increased desirable polyphenols such as chlorogenic acid （CGA） and rutin. In this study, the effects of overexpression of both phenylalanine ammonia lyase （AtPAL2）, the first enzyme of the phenylpropanoid pathway, and hydroxycinnamoyl-CoA quinate：hydroxycinnamoyl transferase （NtHQT）, the last enzyme of CGA biosynthesis, and the overexpression of AtPAL2 together with silencing of NtHQT were investigated in tobacco. Transgenic tobacco plants overexpressing AtPAL2 showed two and five times increases of CGA and rutin levels than the wild-type （WT） plants, respectively. Overexpression of NtHQT further increases the accumulation of CGA in the AtPAL2 plants to about three times than that of the WT level, while silencing of NtHQT in AtPAL2 plants results in -12 times increase in rutin level than that of the WT plants. Simultaneous overexpression of phenylalanine ammonia lyase （PAL） and overexpression/silencing HQT could be used for the production of functional food with increased polyphenols.     

Action de molecules a proprietes hormonales sur l'activite phenylalanine ammoniac lyase

A comparative study has been made of phenylalanine ammonia lyase (PAL) activity in plants sensitive or resistant to various herbicides (piclorame, methylchloro-phenoxyacetic acid (MCPA), atrazine). Piclorame, a herbicide with hormonal activity caused a large decrease in PAL activity of sensitive plants (Nicotiana tabacum), even at low concentrations (5 × 10^-9M) whilst in resistant plants (Triticum aestivum) its effect is negligible; MCPA, also a herbicide with hormonal activity, similarly affects the activity of PAL, but only at higher concentrations. On the contrary, the action of atrazine, which has no hormonal activity, is lower and weaker, probably being only a secondary effect. Determinations of PAL activity during the photoperiod following piclorame application indicate that this herbicide influences principally the photodependent enzyme activity.     

On the role of flavonoids in the integrated mechanisms of response of Ligustrum vulgare and Phillyrea latifolia to high solar radiation

The role of flavonoids in mechanisms of acclimation to high solar radiation was analysed in Ligustrum vulgare and Phillyrea latifolia, two Mediterranean shrubs that have the same flavonoid composition but differ strikingly in their leaf morpho-anatomical traits. In plants exposed to 12 or 100% solar radiation, measurements were made for surface morphology and leaf anatomy; optical properties, photosynthetic pigments, and photosystem II efficiency; antioxidant enzymes, lipid peroxidation and phenylalanine ammonia lyase; synthesis of hydroxycinnamates and flavonoids; and the tissue-specific distribution of flavonoid aglycones and ortho-dihydroxylated B-ring flavonoid glycosides. A denser indumentum of glandular trichomes, coupled with both a thicker cuticle and a larger amount of cuticular flavonoids, allowed P. latifolia to prevent highly damaging solar wavelengths from reaching sensitive targets to a greater degree than L. vulgare. Antioxidant enzymes in P. latifolia were also more effective in countering light-induced oxidative load than those in L. vulgare. Consistently, light-induced accumulation of flavonoids in L. vulgare, particularly ortho-dihydroxylated flavonoids in the leaf mesophyll, greatly exceeded that in P. latifolia. We conclude that the accumulation of flavonoid glycosides associated with high solar radiation-induced oxidative stress and, hence, biosynthesis of flavonoids appear to be unrelated to 'tolerance' to high solar radiation in the species examined.     

Biological control of root-knot nematode (Meloidogyne javanica) disease by Pseudomonas fluorescens (Chao)

Plant growth-promoting Rhizobacteria is currently developed as an biocontrol agent against many plant pathogens. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Pseudomonas fluorescens was investigated in greenhouse and laboratory experiments. Results showed that 10⁹?(CFU/ml) of P. fluorescens decreased nematode infection and other parameters significantly, compared to the control. P. fluorescens was able to cause destruction of nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX) and phenylalanine ammonia lyase (PAL), increased significantly in P. fluorescens-inoculated plants. Maximum activities of POX and PAL were observed at the 5?days after inoculation, respectively. Results suggested that the destruction of eggs and plant defence mechanisms leading to systemic resistance are two main suppression mechanisms used by P. fluorescens against nematode.     

粘红酵母产L-苯丙氨酸解氨酶发酵培养基的优化

通过单因子和正交试验 ,对粘红酵母产 L -苯丙氨酸解氨酶 ( PAL )培养基进行优化 ,L-苯丙氨酸的积累浓度可以从 2 .0 g/1 0 0 ml提高到 3 .3 g/1 0 0 ml,最终得到了 L-苯丙氨酸解氨酶发酵的最适条件     

Incorporation de phenylalanine,de glucose et d'acide cinnamique dans les flavonols de l'oignon

In Allium cepa bulb scales, incorporation of ¹⁴C-phenylalanine, cinnamic acid and glucose were studied in relation to flavonol synthesis. The best incorporation into flavonols is obtained with either cinnamic acid or phenylalanine. ¹⁴C-glucose gives a slow incorporation into flavonol aglycone; this is because there is a big pool of free glucose in the scales in which the precursor is diluted. Under certain conditions, free cinnamic acid is quickly incorporated in a complex which may be a glycoside. After short labelling experiments with phenylalanine or cinnamic acid, some free precursor can be found in the scales a few days later but it is not available for flavonol synthesis. In these conditions, flavonol analysis shows in some cases, no turnover and in others, a turnover of 10% per day due to catabolism.