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.     

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.     

Phenylalanine ammonia-lyase activity in callus cultures of Pinus elliotii

Phenylalanine ammonia-lyase (PAL) activity increased 8- to 12-fold in pine (Pinus elliotii Engelm.) callus tissue within 2 days after subculturing on fresh medium. Factors such as increasing the sucrose content of the media, imposing additional tissue in jury or subculturing more frequently did not cause additional stimulation of PAL activity. The rapid increase in PAL activity appeared to be due to enzyme activation, since cycloheximide did not appreciably reduce the stimulation of PAL activity. The subsequent loss of increased PAL activity with age was reduced by cycloheximide and a cool growth environment.     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of chitin hexamer and thuricin 17 on lignification-related and antioxidative enzymes in Soybean Plants

Inducers of disease resistance in crop plants have a role in sustainable agriculture. We describe a set of bacteriocins that can potentially improve plant growth by controlling specific pathogens and inducing generalized resistance. Solutions of the bacteriocin thuricin 17 and/or a chitin hexamer (a known inducer and positive control) were applied to leaves of two-week-old soybean plants, and levels of lignification-related and antioxidative enzymes were monitored. Phenyl ammonia lyase (PAL) activity in thuricin 17-treated leaves was highest at 60 h after treatment, being 61.8% greater than the control. PAL activity also was increased 18.1% at 72 h after treatment with the chitin hexamer. Tyrosine ammonia lyase (TAL) activity in leaves was 57.0% higher than the control at 48 h after treatment with thuricin 17, while such activity in chitin hexamer-treated leaves was increased by 23.8% at 72 h. At 36 h after treatment with the chitin hexamer or chitin hexamer + thuricin 17, the total concentration of phenolic compounds was 15.3 or 19.3%, respectively, greater than the control. At 72 h, total phenolic concentrations increased by 23.2 and 19%, respectively, in response to thuricin 17 and chitin hexamer + thuricin 17. POD activity in thuricin 17-treated leaves increased by 74.6 and 81.2% at 48 and 72 h, respectively, whereas SOD activity increased by 24.9 and 79.9%, respectively, in chitin hexamer- and thuricin 17-treated leaves at 48 h. A peroxidase isozyme (31 kDa isomer) was induced in thuricin 17-treated leaves at 60 h, while catalase (59 kDa isomer) was induced in chitin hexamer-treated leaves. PAGE showed that two major SOD bands (Fe-SODs) were produced by both types of treatment. Collectively, these results indicate that the bacteriocin thuricin 17 can act as an inducer of plant disease defenses (i.e., activated lignification-related enzymes, antioxidative enzymes, and related isozymes) and that this induction is similar, but not identical, to that of the chitin hexamer elicitor. Although treatment with thuricin 17 + chitin hexamer also induced those responses, it did not present a clear pattern of additivity or synergy.     

Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi

Biological production of p-hydroxycinnamic acid (pHCA) from glucose can be achieved via deamination of the aromatic amino acids l-tyrosine or l-phenylalanine. Deamination of l-phenylalanine produces trans-cinnamic acid (CA) which is further hydroxylated in the para position to produce pHCA. However, when tyrosine is used as the substrate, trans-pHCA is produced in one step. This reaction is accomplished by phenylalanine ammonia-lyase (PAL)/tyrosine ammonia-lyase (TAL). Various bacteria and eukaryotic microorganisms were screened for their ability to produce a PAL/TAL enzyme with high TAL activity. Cell-free extracts of the yeast Rhodotorula glutinis possessed the highest level of TAL activity (0.0143U/mg protein) and the lowest PAL/TAL ratio (1.68) amongst species examined. The gene for this enzyme was cloned and expressed in Escherichia coli and the kinetics of the purified PAL/TAL determined. The recombinant PAL/TAL possessed characteristics similar to those of the wild-type enzyme. Functional expression of R. glutinis PAL/TAL enzyme in Saccharomyces cerevisiae cells containing the plant C4H P-450 and P-450 reductase enzymes from Helianthus tuberosus allowed conversion of glucose to pHCA. Addition of l-phenylalanine to these cultures increased pHCA production confirming its production via the PAL route. When R. glutinis PAL/TAL was synthesized in an E. colil-phenylalanine producing strain (ATCC 31882) and grown on glucose, pHCA was formed in the absence of the Cytochrome P-450 and the P-450 reductase enzymes underlining its production via the TAL route without CA intermediacy.     

Regulation of induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

Total RNA was extracted from fast growing suspension cells of bean, the mRNA was translated and the products of protein synthesis analysed by gel electrophoresis. Actinomycin D (20 g ml^–1) added to the cultures 12 h before the induction of phenylalanine ammonia-lyase (PAL) activity by naphthylacetic acid (NAA) (1 mg/l) and kinetin (0.2 mg/l) failed to prevent the increased activity of the enzyme usually produced by this ratio of the plant growth hormones. PAL was isolated and purified from suspension cultured bean cells. The purified enzyme ran as a single band on polyacrylamide gel electrophoresis. The protein translated from RNA prepared from induced and non-induced cells was separated by gel electrophoresis and the bands of protein on the gels were compared. There was no evidence for an increase in the amount of PAL synthesised in vitro from the mRNA of induced cells even though these had 5 times the amount of activity of the enzyme compared with that of the non-induced cells. The results indicate that the induction of PAL activity is not immediately preceeded by an increase in the synthesis of PAL-mRNA by the cells. The control of the activity of the enzyme is discussed with respect to this finding.Abbreviations PAL phenylalanine ammonia-lyase - NAA 3naphthylacetic acid - DEAE Diethylamino ethyl - EDTA Ethylenediamine tetraacetate - SDS Sodium dodecyl sulphate     

Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots

The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.     

Phenylalanine ammonia-lyase-mediated biosynthesis of 2-hydroxy-4-methoxybenzaldehyde in roots of Hemidesmus indicus

The fragrant rootstocks of Hemidesmus indicus are known to accumulate 2-hydroxy-4-methoxybenzaldehyde (MBALD), yet, the enzymatic route to this hydroxybenzoate is not known. Therefore, root organs of H. indicus hold promises to unravel the biosynthesis related to this phenolic fragrance. Chitosan treatment at 200mg/L concentration to the excised roots effectively increased phenolic accumulation in both the cortex and cork tissues reaching a peak after 24h treatment and decreasing thereafter. The activity of phenylalanine ammonia-lyase (PAL) enzyme in excised roots also increased upon chitosan elicitation, and the maximum specific activity was recorded after 12h of elicitation. Suppression of PAL in vivo by using a specific irreversible inhibitor aminooxyacetic acid (AOAA) resulted in the decrease in MBALD content, indicating its formation via phenylpropanoid pathway.     

The effect of calcium deficiency on the activity of ammonia-lyases in the shikimic pathway

Calcium deficiency resulted in a 20% increase in the total content of phenolic compounds in the shoots of 10-day-old wheat plants and in a 10% decrease in the roots compared with the control. Differences in specific activities of the lyases of the shikimate pathway (L-phenylalanine ammonia-lyase, E.C.4.3.1.5, PAL; L-tyrosine ammonia-lyase, TAL) were established between control and Ca-deficient wheat and pumpkin plants. The lyase activities were higher in the shoots of Ca-deficient plants and lower in their roots, compared with the control. Thus, there is a certain correlation between PAL and TAL activities on the one hand and the content of phenolic compounds on the other. The time course of PAL activity during a few first days of growth and the dependence of TAL activity on pH were also investigated.     

Immunochemical localization of phenylalanine ammonia-lyase and chalcone synthase in anthers

Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) from anthers of the garden tulip Apeldoorn have been purified to apparent homogeneity as revealed by sodium dodecyl sulfate disc-gel electrophoresis. Phenylalanine ammonia-lyase was either purified by successive chromatography on Sephacryl S 300 Superfine, HA Ultrogel and on diethylaminoethyl Sephacel or by immunoaffinity chromatography in a single step. Purification of CHS was achieved by chromatography on Sephadex G 200 and on HA Ultrogel followed by chromatofocusing. The purified enzymes were used for the immunization of rabbits. The specificity of the antisera against both PAL and CHS was tested by diverse methods. Antisera against PAL and CHS were employed to detect the localization of the enzymes in cross sections of tulip anthers using an indirect immunofluorometric method. The results show that PAL and CHS are located predominantly in the tapetum cells. These observations strengthen the view that the tapetum plays an important role in the regulation of phenylpropanoid metabolism within the loculus of anthers.Abbreviations CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS sodium dodecyl sulfate Some of the results were presented at the meeting of German Botanical Society in Freiburg, FRG, September 1982, and at the meeting of the Groupe Polyphenols in Toulouse, France, September/October 1982     

Modulation of L-phenylalanine ammonia-lyase by pathway intermediates in cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.)

The increase in extractable phenylalanine ammonia-lyase (PAL;EC 4.3.1.5.) activity induced in French bean cell suspension cultures in response to treatment with autoclaved ribonuclease A was inhibited by addition of the phenylpropanoid pathway intermediates cinnamic acid, 4-coumaric acid or ferulic acid. The effectiveness of inhibition was in the order cinnamic acid>4-coumaric acid>ferulic acid. Cinnamic acid also inhibited the PAL activity increase induced by dilution of the suspensions into an excess of fresh culture medium. Addition of low concentrations (<10^-5M) of the pathway intermediates to cultures at the time of application of ribonuclease gave variable responses ranging from inhibition to 30–40% stimulation of the PAL activity measured at 8 h. Following addition of pathway intermediates to cultures 4–5 h after ribonuclease treatment, rapid increases followed by equally rapid declines in PAL activity were observed. The cinnamic acid-stimulated increase in enzyme activity was unaffected by treatment with cycloheximide at a concentration which gave complete inhibition of the ribonuclease-induced response. However, cycloheximide completely abolished the subsequent decline in enzyme activity. Treatment of induced cultures with -aminooxy--phenylpropionic acid (AOPPA) resulted in increased but delayed rates of enzyme appearance when compared to controls not treated with the phenylalanine analogue. The results are discussed in relation to current views on the regulation of enzyme levels in higher plants.Abbreviations AOPPA -aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - AOA -aminooxyacetic acid     

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     

<Emphasis Type="Italic">Lyso</Emphasis>-phosphatidylethanolamine-enhanced phenylalanine ammonia-lyase and insoluble acid invertase in isolated radish cotyledons

Application of lyso-phosphatidylethanolamine (LPE) is purported to suppress fruit ripening and delay foliar senescence. However, the endogenous LPE response of plants is more typically associated with propagation of wound and stress signals. Experiments were therefore carried out to determine whether exogenous LPE could elicit defense responses in plants by determining the effect of this lyso-phospholipid on activity of two key metabolic enzymes and pathogenesis-related proteins viz phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and insoluble acid invertase (Ac INV; EC 3.2.1.26) in expanding cotyledons of Raphanus sativus L. cv. Cherry Belle (radish). Activity of both enzymes was increased following exposure of tissue to 18:0-LPE and the response was dose dependent. Soluble Ac INV activity was not enhanced by exogenous 18:0-LPE. Increased PAL activity appeared to coincide with a decline in phenolic acid content and a rise in sinapine and lignin. An increase in insoluble Ac INV by 18:0-LPE was associated with a reduction in sucrose concentration. However, levels of glucose and fructose were unaffected. In view of these findings it is proposed that applied LPE acts to co-ordinate carbohydrate partitioning locally to fulfill anabolic respiratory requirements associated with the propagation of systemic wound and stress responses. Furthermore, the impact of exogenous 18:0-LPE on insoluble Ac INV activity is discussed in relation to the proposed role of this enzyme in cytokinin-mediated senescence delay.     

异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

以异叶天南星(Arisaema heterophyllum Blume)为材料,采用逆转录PCR(RT-PCR)法扩增其苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)基因Ah PAL,获得该基因的开放读码框(ORF),并通过系统的生物信息学软件分析Ah PAL的结构和理化性质。结果显示,Ah PAL的ORF全长为2184 bp,编码727个氨基酸;Ah PAL与郁金香(Tulipa fosteriana W. Irving) PAL的亲缘关系最近,序列相似性达88%。空间结构模型分析结果显示,Ah PAL为同型四聚体,每个单体由3个结构域组成,其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心,是Ah PAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个Ah PAL Unigene在根、块茎和叶中的表达情况,发现它们在根中表达量均最高,而在叶和块茎中表达较低。     

异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

以异叶天南星（Arisaema heterophyllum Blume）为材料,采用逆转录PCR（RT-PCR）法扩增其苯丙氨酸解氨酶（Phenylalanine ammonia-lyase,PAL）基因AhPAL,获得该基因的开放读码框（ORF）,并通过系统的生物信息学软件分析AhPAL的结构和理化性质。结果显示,AhPAL的ORF全长为2184 bp,编码727个氨基酸;AhPAL与郁金香（Tulipa fosteriana W.Irving）PAL的亲缘关系最近,序列相似性达88%。空间结构模型分析结果显示,AhPAL为同型四聚体,每个单体由3个结构域组成,其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心,是AhPAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个AhPAL Unigene在根、块茎和叶中的表达情况,发现它们在根中表达量均最高,而在叶和块茎中表达较低。