Search

Background The application of arbuscular mycorrhizal fungi (AMF) can induce the synthesis and accumulation of secondary metabolites in the tissues of host plants, thus impacting their allelopathic potential. Materials and methods The objective of this study was to determine the effect of three AMF species (Rhizoglomus intraradices, Funneliformis mosseae, Rhizoglomus fasciculatum) on photosynthetic pigments and secondary metabolites content in roots and reproductive organs of Ipomoea purpurea L., Digitaria sanguinalis L., and Solanum nigrum L. as a problematic weed species. Results Among compared weeds, the roots of D. sanguinalis associated with AMF accumulated the highest level of phenols. Higher content of flavonoids was obtained in roots of S. nigrum (7.46 mg g−1 FW) following colonization with R. intraradices. Berries of S. nigrum inoculated with R. intraradices had a higher concentration of terpenoids (21.45 mg 100 mL−1 of extract) than reproductive organs of D. sanguinalis and I. purpurea. Colonization with R. intraradices improved total phenolics in seeds of D. sanguinalis compared to the reproductive organs of other weeds. These compounds released from seeds help defend against pathogen infection, consequently increasing seed production. In addition, phenylalanine ammonia lyase enzyme activity in leaves of D. sanguinalis colonized by R. fasciculatum and F. mosseae was 55% and 67%, respectively, higher than I. purpurea plants, grown in the same condition. Conclusions Results suggest that AMF can play a crucial role in enhancing of secondary metabolites in these three weeds, thereby improving their allelopathic potential and competitive ability. Graphic Abstract

We investigated changes in the expression of Tmpal1 and Tmpal2, that encode phenylalanine ammonia-lyases (PALs), during the growth of Tricholoma matsutake NBRC 30605 mycelia, and the effect of exogenously added l -phenylalanine ( l -Phe), a substrate of PALs, on pal expression and methyl cinnamate production in the mycelia. Expression patterns of Tmpal1 and Tmpal2 differed during mycelial growth. Transcript levels of Tmpal2, PAL activity, and methyl cinnamate content increased markedly in cultures supplemented with 4–6 mM l -Phe. These results indicate that l -Phe supplementation increases PAL activity that is mainly responsible for Tmpal2 expression; consequently, methyl cinnamate is abundantly produced from l -Phe.

The effect of temperature on phenolic accumulation in fresh-cut pitaya fruit was reported during storage at 5, 10 and 15 °C. Fresh-cut pitaya fruit were treated with ROS production inhibitor (diphenyliodonium iodide, DPI) or elicitor (glucose/glucose oxidase, G/GO) and the total soluble phenolics (TSP) content, antioxidant activity (AOX), phenylalanine ammonia lyase (PAL) activity, as well as ROS production and antioxidant enzymes activity were determined. Results showed that higher storage temperature promoted the production of ROS, enhanced the accumulation of TSP and improved the AOX of fresh-cut pitaya fruit. Moreover, treatment with DPI dramatically inhibited ROS generation, PAL activity and TSP accumulation, while G/GO treatment significantly increased ROS level, PAL activity and TSP accumulation. These results demonstrate that phenolics accumulate with increasing temperature and confirm previous report of ROS effects on wound induced phenolic accumulation in pitaya fruit.

The single and combined effects of UV-B (1.5 kJ m −2 ) and UV-C (4.0 kJ m −2 ) radiation treatments were studied on the phenylalanine ammonia-lyase (PAL) activity, phenolic content and antioxidant capacity (TAC) of shredded fresh-cut carrots during 72 h at 15 °C. Non-irradiated samples were used as control (CTRL). PAL activity of UV-B and CTRL samples was increased by approximately 760% after 72 h while increases of 120–140% in the remaining radiation treatments were found. Chlorogenic acid represented 70% of the sum of phenolic compounds of initial samples. UV-B treatment showed the highest phenolic accumulation with 498% after 72 h, regarding its initial value, while single and combined UV-C treatments achieved an accumulation of 440%, which was similar to CTRL samples. Such phenolics data were highly correlated (R 2 = 0.82) to total antioxidant capacity throughout storage. Conclusively, UV-B treatment achieved the highest phenolic compounds accumulation and related antioxidant capacity in shredded carrot during 3 days at 15 °C. Furthermore, the use of UV-C treatment with sanitizing purposes combined with UV-B radiation did not negatively affect the accumulation of bioactive compounds, achieving similar enhancements to CTRL samples.

Inhibition of browning in gamma irradiated (2 kGy) RTC ash gourd during storage (10 °C, 12 days) was investigated. Irradiation resulted in a significant increase in the content of α-resorcylic acid that was correlated with decreased PPO activity. Nature of the PPO inhibition was found to be of mixed and reversible type with K i , 0.09 mM; K is , 27.33 mM and IC 50 of 25 mM. During storage, the non-irradiated control samples exhibited increased oxidation of leached phenolic compounds at the surface as a consequence of changes in tissue micro-structure compared to the irradiated samples. Unlike control samples that exhibited enhanced PAL activity, quinone formation and browning, no increase in PAL activity and browning was noted in the irradiated samples during storage. Browning inhibition in stored radiation-processed (2 kGy) RTC ash gourd was thus proposed to be a synergistic effect of decreased quinone formation and reduced enzyme (PPO and PAL) activities. Industrial relevance The present study demonstrates the feasibility of gamma irradiation for browning inhibition in ready-to-cook (RTC) ash gourd during storage. Use of gamma irradiation for optimizing the post-processing conditions for improved visual appeal and storability of the fresh-cut produce in food industry is thus recommended. The possible use of alpha resorcylic acid as a natural browning inhibitor in fruits and vegetables is also of industrial relevance. The various approaches proposed in the study can be utilized by the food industry for maintaining visual quality of fresh-cut produce during storage thereby improving marketability of such produce.

The effect of allyl isothiocyanate (AITC), in combination with low temperature (10°C) storage on post harvest quality of minimally processed shredded cabbage was investigated. An optimum concentration of 0.05μL/mL AITC was found to be effective in maintaining the microbial and sensory quality of the product for a period of 12days. Inhibition of browning was shown to result from a down-regulation (1.4-fold) of phenylalanine ammonia lyase (PAL) gene expression and a consequent decrease in PAL enzyme activity and o-quinone content. In the untreated control samples, PAL activity increased following up-regulation in PAL gene expression that could be linearly correlated with enhanced o-quinone formation and browning. The efficacy of AITC in extending the shelf life of minimally processed shredded cabbage and its role in down-regulation of PAL gene expression resulting in browning inhibition in the product is reported here for the first time.

Previously, we found that phenolic content and antioxidant capacity (AOX) in carrots increased with wounding intensity. It was also reported that UV radiation may trigger the phenylpropanoid metabolism in plant tissues. Here, we determined the combined effect of wounding intensity and UV radiation on phenolic compounds, AOX, and the phenylalanine ammonia-lyase (PAL) activity of carrots. Accordingly, phenolic content, AOX, and PAL activity increased in cut carrots with the duration of UVC radiation, whereas whole carrots showed no increase. Carrot pies showed a higher increase compared to slices and shreds. Phenolics, AOX, and PAL activity also increased in cut carrots exposed to UVA or UVB. The major phenolics were chlorogenic acid and its isomers, ferulic acid, and isocoumarin. The type of UV radiation affected phenolic profiles. Chlorogenic acid was induced by all UV radiations but mostly by UVB and UVC, ferulic acid was induced by all UV lights to comparable levels, while isocoumarin and 4,5-diCQA was induced mainly by UVB and UVC compared to UVA. In general, total phenolics correlated linearly with AOX for all treatments. A reactive oxygen species (ROS) mediated hypothetical mechanism explaining the synergistic effect of wounding and different UV radiation stresses on phenolics accumulation in plants is herein proposed.

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1 mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1 mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5 mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1 mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8 μg ml(-1) for the HL-60, HeLa, and MCF-7 cells, respectively.

Interactions between jasmonic acid (JA) and salicylic acid (SA) signaling pathways play important roles in the regulation and fine-tuning of induced defenses which are activated by pathogen, insect attack, or wounding. In this study, endogenous SA and JA level in pea seedlings presented opposite trends in response to wounding. Results exhibited that SA impaired the resistance to wounding of pea seedlings by means of suppressing the action of JA. Based on the fact that the wound-induced PPO activity was suppressed by SA, it could be concluded further that SA had negative effect on JA-related defense response. However, synergy between JA and SA also existed in phenolic metabolism related to PAL. Moreover, endogenous SA and salicylic acid 2-O-β- d -glucose (SAG) levels in wounded pea seedlings also appeared opposite variation tendency, and newly synthesised SAG was detected. In contrast to SA, SAG showed some synergistic action in JA-related defense response in terms of MDA, PPO activity and PAL activity, which was quite different from SA. In conclusion, these results revealed both the negative effect and some synergistic effect of SA on JA-related defense of pea in response to wounding, particularly the role of SAG.

Increasing evidence suggests that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is associated with low temperature stress in plant tissues. Banana fruit are highly susceptible to chilling injury. However, little is known about the role of PAL (i.e. gene expression, protein level and activity) in fruit chilling. In this work, the involvement of PAL induced by heat treatment (38 degrees C for 3 days) prior to storage (8 degrees C) in chilling tolerance was investigated. The PAL inhibitor 2-aminoindan-2-phosphonic acid (AIP) was also used to further study the role of PAL in the chilling tolerance. The results showed that mRNA transcripts (MaPAL1 and MaPAL2) and PAL protein levels increased during storage at chilling temperature. Heat treatment prior to storage alleviated chilling injury and enhanced PAL activity, protein amount and MaPAL1 and MaPAL2 transcript levels. The increases in parameters of PAL upon heat pretreatment were all inhibited by AIP treatment, which resulted in aggravation of chilling injury. Thus, these findings indicate that the induction of PAL by heat pretreatment was regulated at both the transcriptional and the translational levels and that PAL may play a role in heat pretreatment-induced chilling tolerance of banana fruit.

Phenylalanine ammonia-lyase activity (PAL, EC 4.3.1.5), total phenolics, soluble proteins, malondialdehyde and metals accumulation in four-week old chamomile (Matricaria chamomilla) plants cultivated in nutrient solution and exposed to low (3 μM) and high (60 and 120 μM) levels of cadmium (Cd) or copper (Cu) for 7 days were studied. High Cd concentrations had a stimulatory effect on PAL activity and soluble phenolics accumulation while high Cu doses decreased soluble proteins in the leaf rosettes. In the roots, extreme stimulatory effects of 60 and 120 μM Cu were observed on PAL activity, phenolics and malondialdehyde accumulation, while protein content was reduced by these Cu doses. Cd accumulation was higher in the leaf rosettes compared to copper, but the opposite was recorded in the roots. Taken together, the stimulatory effect of Cu on phenolic metabolism was recorded, even though high malondialdehyde accumulation may be an indication that phenolics was not sufficient to counteract reactive oxygen species formation thus leading to damage of membrane integrity. In comparison to Cd, Cu had more noticeable effect on the parameters studied to support its strong redox-active properties. These facts in correlation to antioxidative properties of phenolic metabolites are also discussed.

The effect of La on flavonoids, chlorophyll contents, and phenylalanine ammonia-lyase (PAL) activity in soybean seedlings under supplementary ultraviolet-B radiation (UV-B, 280 ∼ 320 nm) was studied. The results show that PAL activity, contents of flavonoids and chlorophyll in the plants pretreated with La (20 mg·L−1) are higher than those in CK. UV-B radiation could result in an increase in flavonoid content and PAL activity, associated with a decrease in chlorophyll content. However, the increase in the range of PAL activity and flavonoid content in UV-B treatment are lesser than those in the La treatment. The changes of flavonoid contents and PAL activity in La + UV-B treatment are similar to those in UV-B treatment, and the increase in their range is higher than those in UV-B treatment. This shows that La can enhance the resistance of soybean seedling to UV-B radiation and alleviate the damage of UV-B radiation by increasing flavonoid content, chlorophyll content, and PAL activity.

Foliar spray and micro-injection of plant growth-promoting rhizobacterial species, viz. Pseudomonas fluorescens and P. aeruginosa on chickpea induced synthesis of phenylalanine ammonia-lyase (PAL) when tested against Sclerotinia sclerotiorum. Induction of PAL was also associated with increased synthesis of phenolic compounds such as tannic, gallic, caffeic, chlorogenic and cinnamic acids. Treatment with P. fluorescens was found to be more effective in inducing phenolic compounds as compared to P. aeruginosa. However, persistence of PAL activity was observed more with P. aeruginosa. Although both the inoculation methods were effective, foliar application was found to be superior to micro-injection in terms of rapid PAL activity leading to the synthesis of phenolic compounds.

Wounding lettuce leaves induces the de novo synthesis of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the accumulation of phenolic compounds, and subsequent tissue browning. A brief heat-shock at 45degreesC reduces the rise in wound-induced PAL, the accumulation of phenolic compounds, and tissue browning. The activity of PAL measured 24 h after wounding and the content of phenolic compounds (absorbance of methanol extract at 320 nm) measured 48 h after wounding was highly correlated (R-2 > 0.90) in tissue developing the normal wound response and in tissue subjected to 0-180 s of heat-shock after wounding. The synthesis of a unique set of proteins called heat-shock proteins (hsps) is induced by these heat-shock treatments. Western-blot analyses of proteins isolated from wounded and heat-shocked Iceberg and Romaine lettuce mid-rib leaf tissue was done using antibodies against hsp 23. Only those heat-shock treatments that were effective at inducing the synthesis of hsp 23 were effective in reducing the activity of PAL induced by wounding and the subsequent accumulation of phenolic compounds. Hsps induced in non-wounded, whole leaves by exposure to 45degreesC for 150 s did not significantly interact with PAL previously synthesized in non-heat-shocked wounded leaves to limit its activity. The preferential synthesis of hsps over that of wound-induced PAL, rather than the presence of hsps, may be responsible for the ability of a heat-shock treatment to reduce the wound-induced increase in PAL activity. Our results support this novel concept, and the possibility that heat-shock treatments can have significant physiological effects on the response of the tissue to other stresses, not because of the specific genes they induce or repress, or the products they cause to be synthesized, but by their secondary action of influencing the synthesis of other proteins (e.g. PAL) by the suppression of non-hsps protein synthesis.

A fluorescent probe (2a-LP) based on an unnatural amino acid (UAA) is developed for the detection of phenylalanine ammonia lyase (PAL). In the presence of PAL, 2a-LP is catalytically deaminated to ortho-amino-transcinnamic acid (o-a-CA), which shows a remarkable “off–on” fluorescence signal. Thus, the probe 2a-LP enables direct visualization of the PAL activity in tomato under UV illumination and has potential in vitro assays.

Wounds and injuries incurred during preparation of fresh-cut lettuce (Lactuca sativa L.) stimulate phenolic metabolism, which leads to tissue browning. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5.) is the first committed enzyme in the phenylpropanoid pathway and regulates its overall activity. Maximum activity of wound-induced PAL occurred sooner as the storage temperature increased from 0 to 25 °C, but the maximums were lower. A heat shock at 50 °C for 90 seconds protected fresh-cut lettuce tissue against browning, helped retain greenness, and decreased subsequent production of phenolics when applied either after or before wounding. Browning was reduced when the heat shock was applied up to 36 hours after wounding, while the maximum effect occurs around 6 hours before cutting. Like the heat-shock treatment, the protein synthesis inhibitor cycloheximide reduced wound-induced PAL activity, but it did not prevent tissue browning. When cycloheximide was applied in combination with heat-shock treatments, browning did not occur. Heat shocks may control tissue browning by more than just interfering with protein synthesis.

The effects of different periods of heating at 37 degrees C on phenylalanine ammonia-lyase (PAL) and how this relates to chilling tolerance was investigated in fruits of the chilling-sensitive Fortune mandarin. All effective heat-conditioning treatments caused an early and transient increase in PAL mRNA and PAL activity. Conditioning fruits at 37 degrees C for 1 or 2 days prevented the manifestation of chilling symptoms but not the accumulation of PAL mRNA and PAL activity observed in untreated fruits. In fruits conditioned for 3 days, cold-induced damage and PAL activity were also suppressed but not the accumulation of PAL transcript upon subsequent storage at 2 degrees C. Storage of 3-day-heated fruits at a nonchilling temperature (12 degrees C) induced an early and transient increase in both PAL mRNA and PAL activity. High levels of PAL transcript and PAL activity were detected in freshly harvested fruits of a chilling-resistant mandarin (Hernandina) that decreased upon cold storage at 2 degrees C in heat-treated and nontreated fruits. These results indicate that sensitivity of mandarins to chilling correlates with low constitutive levels of PAL mRNA and PAL activity and with the inducibility of both upon exposure to low temperatures.

Cell suspension cultures were generated from transgenic tobacco plants expressing bean phenylalanine ammonia-lyase (PAL) or alfalfa cinnamate 4-hydroxylase (C4H) transgenes. PAL over-expressing cell lines exhibited high basal levels of PAL transcripts and enzymatic activity, but hardly exceeded wild-type cultures in the level of PAL activity obtained following elicitation. In contrast, expression of alfalfa C4H transcripts in unelicited cell suspension cultures did not result in a corresponding increase in C4H enzymatic activity. However, following elicitation, the level of C4H activity in over-expressing lines was significantly greater than in control lines.

Phenylpropanoid compounds encompass a wide range of structural classes and biological functions. Limiting discussion to stress-induced phenylpropanoids eliminates few of the structural classes, because many compounds thst are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant (Beggs et al., 1987; Christie et al., 1994).

Summary PAL activity in potato tuber disks was markedly increased during the course of incubation. The amounts of ethylene from the disks were very low. Removal of ethylene from the disks slightly stimulated the increase in PAL activity while the addition of ethylene suppressed it. These results are in contrast with other system in which the development of PAL was greatly stimulated by ethylene.

A factor capable of the reversible inactivation of PAL in vitro has been demonstrated in extracts of gherkin hypocotyls. Kinetics of the interaction between enzyme and inactivator indicate that PAL and the factor form a freely reversible complex. The properties of the inactivator are discussed in relation to its proposed role in the regulation of PAL activity in dark- and light-grown tissue.

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

Phenylalanine ammonia-lyase (PAL) activity is almost nil in cotyledons of intact seedlings 48 h old, but rapidly increases in the 3 h following the excision. Moreover, activity in excised cotyledons appears to depend on the surface in contact with air, indicating that the processes leading to PAL activity increase are oxygen-dependent. Treatment with fusicoccin (FC) causes a remarkable increase in fresh weight, irrespective of whether treatment is carried out in light or in darkness. FC is therefore effective in inducing cotyledon enlargement regardless of light conditions. The effect of light on PAL is nearly identical in cotyledons incubated in water or in FC, while FC has no effect with respect to water, in darkness as well as in light. Incubation in darkness in kinetin 10−4 M causes a fresh weight increase which is about twice that of controls in water, while kinetin has still no effect on PAL activity. Our data show that FC and kinetin have no significant effect on PAL activity in excised cotyledons, while they stimulate enlargement. This suggests that light acts on PAL more specifically than through cotyledon enlargement.

Application of L-α-aminooxy-β-phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283-287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.

Summary The photoregulation of l -Phenylalanine ammonia-lyase (PAL) is studied by immunochemical methods. We used a partly purified l -Phenylalanine ammonia-lyase: F1 light fraction, the corresponding inactive one provided from dark-grown cotyledons: F1 dark fraction and the antisera specific of these two fractions. The complete absorption of PAL activity from F1 light fraction with the anti-F1 light immune serum shows the antigenicity of PAL and the specificity of this serum for all forms of PAL present in F1 light fraction. The presence of an inactive l -Phenylalanine ammonia-lyase in the 36 h dark-grown cotyledons suggested by preliminary results of absorption is confirmed by showing that less PAL activity is precipitated from the fraction F1 light by a same amount of IgG anti-F1 light when F1 dark fraction is added. This result is explained by a competition between active and inactive forms of PAL for the IgG extracted from an immune serum specific for F1 light fraction. By measuring the absorption of PAL activity from F0 fraction (crude extract) obtained from 18 h, 36 h and 48 h light-grown cotyledons when increasing amounts of IgG anti F1 36 h light are added, we demonstrate the presence of at least two isozymes A and B, the synthesis of B being shifted in time in comparison to A.

The exposure of potatoes to gamma rays (10 krad) caused on induction of phenylalanine ammonia lyase (PAL) in the tubers. The induced activity was localized essentially in cortex tissue, mainly concentrated around the bud region. Whereas in parenchymatous tissue no induction was observed. The enhancement in PAL activity in buds was observed to be dependent on dose of irradiation and the duration of storage thereafter. The tubers exposed to a 10 krad dose showed maximal activation on storage at ambient conditions with 20 fold activity observed within three hours after irradiation. On further storage the activity declined and reached a constant level within a period of one week. The increase in PAL activity was partly due to activation of the inactive form of the enzyme (30–40%) and partly due to de nova synthesis of the protein (60–70%).

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.

Summary In castor beans maximum activities of tyrosine ammonia lyase (TAL) and phenylalanine ammonia lyase (PAL) in the endosperm were found at day 4 of germination. Wounding caused a strong increase in PAL activity but did not affect TAL. Treatment of the seedlings with MnCl 2 drastically enhanced PAL activity, but only a slight increase of TAL was observed. DEAE cellulose chromatography resulted in only one peak of TAL activity, whereas 2 peaks were found for PAL.

Gibberellic acid (10−6 g GA3/ml) present in the medium during 48 hours of irradiation with blue light completely inhibited anthocyanin production inHaplopappus cultures and PAL activity was 60% lower by this treatment. In the dark only a 35% inhibition of the enzyme was found. Compared to untreated controls synthesis of phenylpropane derivates after addition of GA3 showed a 40% inhibition in the blue light irradiated cultures and a 5% lower level in the cultures kept in the dark.

D-Phenylalanine, contrary to its L-stereoisomer causes an increase in L-phenylalanine ammonia-lyase (PAL) activity during the culture of dormant and stratified apple embryos and during the germination of mustard, rape and oat seeds. None of the other six pairs of L- and D-aminoacids tested caused a comparable effect. It was demonstrated that the increase of PAL activity follows the normal germination and that the stimulation of this activity by D-phenylalanine is not directly related either to germination or to the growth processes. The possible mechanism of PAL stimulation in vivo is discussed.

The time-course of phenylalanine ammonia-lyase (PAL) activity and of apparent phytochrome content has been determined under continuous farred irradiation. The kinetics of PAL (EC 4.3.1.5) show four phases: after a lagphase (1) which is detectable only in the early stages of the development, PAL activity increases (2), reaches a maximum (3) and then decreases (4). The development of PAL activity is controlled by the far-red absorbing form of the phytochrome (Pfr) through an all-or-none phenomenon. Whilst phytochrome content is above a critical value ( C m ) PAL activity increased. The lag-phase, the maximum and the decrease of PAL activity can be explained by the fact that the phytochrome content is below the C m .

Induction of phenylalanine ammonia lyase (PAL) by γ-irradiation was tissue-specific and maximum activity was obtained in bud tissue. Parenchyma tissue which showed maximum induction with light did not respond to γ-rays. Exposure of excised unirradiated bud tissue to light did not show any lag period in the induction of PAL. On the other hand, bud tissue excised from irradiated potatoes on exposure to light showed 4 h delay in further induction of PAL. The induction of PAL in parenchyma tissue was delayed by about 4 h. Reirradiation of the tissue 3 h after irradiation could not induce more PAL activity. Similarly tissues exposed to light responded to γ-irradiation only after 24 h when light-induced PAL activity started to decline. Light-induced and irradiation-induced enzymes from bud tissue showed identical properties such as optimum pH (8.8), Km (70 μM), Ki for cinnamic acid (70 μM) and electrophoretic mobility.

Ethylene stimulated the activity of l-phenylalanine ammonia-lyase (PAL) in carrot (Daucus carota L.) tissues. It is also known to induce the formation of isochlorogenic acid. However, the induction patterns were different for isochlorogenic acid and PAL activity. Ethylene action seems to be indirect, as it did not affect the PAL activity of cell-free extracts.

Exogenous supplies of phenylalanne, cinnamic acid and p-coumaric acid can inhibit the appearance of phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) activity in potato tuber discs, and exogenous supplies of cinnamic acid and p-coumaric acid can inhibit the appearance of cinnamic acid 4-hydroxylase (CA4H, E.C. 1.14.13.11) activity. The time-courses of the inhibitory effects of (a) exogenous phenylalanine on the appearance of PAL activity and (b) exogenous cinnamic acid on the appearance of CA4H activity suggest that the respective substrates of the two enzymes have no inhibitory effects per se, but rather that metabolic derivatives are the active agents. Accordingly, cinnamic acid and p-coumaric acid, but not phenylalanine can inhibit the initial appearance of PAL activity. Similarly, p-coumaric acid but not cinnamic acid can inhibit the initial appearance of CA4H activity. The inhibitory effects of exogenous cinnamic acid and p-coumaric acid on the initial appearance of PAL activity are independent. These findings are discussed in relation to the control in vivo of the levels of activity of PAL and CA4H.

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.

Fluctuations in the activity of phenylalanine ammonia-lyase (PAL) in tissues of buckwheat seedlings were monitored by an intact cell assay and compared with results obtained with the conventional in vitro assay. Good correlation between measurements with the different assays was found for PAL activity changes during 1) growth of seedlings in the dark, 2) illumination of seedlings, and 3) incubation of hypocotyl segments. When leaf disks of buckwheat, strawberry, or sunflower were allowed to float on either water or a sugar solution, PAL activities determined with the intact cell assay increased irrespective of the presence of sugar, while PAL activities determined in cell homogenates increased only in disks floating on a sugar solution. Possible explanations for these discrepancies are discussed.

Summary Anaerobiosis, carbon monoxide and azide inhibit the PAL activity development in radish cotyledons. An activity increase can occur with only 3% oxygen after excision while for significant light induction 20% oxygen is necessary.

Cycloheximide inhibits the appearance of phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) activity and cinnamic acid 4-hydroxylase (CA4H) (EC 1.14.13.11) activity in illuminated potato tuber discs, but the appearance of PAL activity is more sensitive to inhibition by low concentrations of cycloheximide than is the appearance of CA4H activity. A differential effect of cycloheximide is also observed in non-illuminated potato tuber discs, but is considerably less marked than in illuminated discs. The decline in PAL activity following a 15 h pre-incubation in an illuminated condition is inhibited by high concentrations of cycloheximide. However, the corresponding decline in CA4H activity is not inhibited by concentrations of cycloheximide that inhibit the decline in PAL activity.

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.

It has been shown that the treatment of intact storage roots of swede and parsnip with low levels of ethylene leads to an increase in respiration and a large rise in the activity of phenylalanine ammonia lyase (PAL). It is shown that in disks of swede aged in the presence of ethylene there is also an increase in the extractable activity of PAL. The increase in PAL activity is very sensitive to the level of ethylene; 1 ppm of ethylene being sufficient to give a half maximal response. IAA at 10-4 M stimulates an increase in endogenous ethylene production of the disks and an increase in PAL activity, but this increase in PAL activity is small in relation to that given by saturating levels of ethylene. It is likely that the ethylene induced increase in PAL activity is due to de novo protein synthesis since it is inhibited by the presence of a range of inhibitors of RNA and protein synthesis. The characteristics of the response are discussed in relation to the other known effects of ethylene.

The literature concerning the physiology and biochemistry of the enzyme phenylalanine ammonia lyase (PAL) (E.C. 4.1.1.5) from different organisms has been reviewed. Levels of the enzyme are affected by age, light, phytochrome, wounding, infection and growth modifiers. The possibility that PAL is involved in the control of phenolic metabolism has been critically examined.

External layers of citrus fruit peel evinced a marked increase in phenylalanine ammonia-lyase (PAL) activity after irradiation with 200 krad doses of gamma radiation. Maximum enzyme activity was attained one day after irradiation. Thereafter PAL activity rapidly decreased to control values in fruits which did not show visible damage symptoms later upon storage, but remained relatively high for several days in those fruits which later showed peel damage. Accumulation of phenolics in cells was demonstrated and may be explained by the enhanced PAL activity. The connections between irradiation, enzyme activity, phenolics and damage are briefly discussed.

Differences in properties of phenylalanine ammonia-lyase (PAL) extracted from the buds of 7-day-old dark-grown pea seedlings and the buds of 7-day-old dark-grown pea seedlings treated with 6 h white light are demonstrated. Attempts to separate these two forms of PAL with normal techniques employed for protein separation have proved negative. The significance of these findings is discussed with reference to current concepts of the regulation of PAL activity.

Changes in phenylalanine ammonia-lyase (PAL) levels in TMV-infected Samsun NN leaves have been investigated in relation to two basic characteristics of hypersensitivity: necrosis and acquired systemic resistance. Higher levels of enzymatic activity are detectable before the symptoms are visible. A sharp increase occurs at the time of appearance and during subsequent development of the local necrotic lesions. The stimulated PAL activity is not related to virus multiplication but to necrosis since it depends markedly on the number of local lesions and since it is localized in narrow rings around the local lesions. In uninoculated leaves PAL activity remains at the preinoculation level during induction and development of systemic resistance. Resistant and nonresistant TMV challenge-inoculated leaves show PAL levels that, are of the same order of magnitude. The relative differences are independent of the degree of resistance and are probably caused by differences in the number of local lesions induced by challenge inoculation. Thus, phenylalanine ammonia-lyase does not seem to be involved in acquired systemic resistance.