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The current study evaluates the role of phenylalanine ammonia-lyase (PAL) and the associated metabolic complex in the accumulation of lignin in common wheat plants ( Tríticum aestívum L.) at the early stages of ontogenesis. The data analysis was performed using plant samples that had reached Phases 4 and 5 on the Feekes scale-these phases are characterized by a transition to the formation of axial (stem) structures in cereal plants. We have shown that the substrate stimulation of PAL with key substrates, such as L-phenylalanine and L-tyrosine, leads to a significant increase in lignin by an average of 20% in experimental plants compared to control plants. In addition, the presence of these compounds in the nutrient medium led to an increase in the number of gene transcripts associated with lignin synthesis ( PAL6 , C4H1 , 4CL1 , C3H1 ). Inhibition was the main tool of the study. Potential competitive inhibitors of PAL were used: the optical isomer of L-phenylalanine-D-phenylalanine-and the hydroxylamine equivalent of phenylalanine-O-Benzylhydroxylamine. As a result, plants incubated on a medium supplemented with O-Benzylhydroxylamine were characterized by reduced PAL activity (almost one third). The lignin content of the cell wall in plants treated with O-Benzylhydroxylamine was almost halved. In contrast, D-phenylalanine did not lead to significant changes in the lignin-associated metabolic complex, and its effect was similar to that of specific substrates.

Enzymatic browning of Iceberg lettuce was studied by subjecting midrib tissues to a series of mild heat treatments. The effects of wounding and subsequent application of a mild heat treatment were examined by monitoring the browning potential (BP) and the activity of three browning-related enzymes (i.e., phenylalanine ammonia lyase [PAL], polyphenol oxidase [PPO], and peroxidase [POD]) during refrigerated storage up to 10 days. Efficient inhibition of browning was achieved by treatment at 50°C for 60 s. The wound-induced increase of the BP and the activity of PAL and POD was effectively suppressed, maintaining their values at initial levels up to 7 days of storage. PPO activity, on the contrary, remained unchanged after wounding, whether or not followed by heat treatment. BP, PAL activity and POD were found to be strongly correlated, whereas meaningful associations for PPO with the other parameters could not be established. PRACTICAL APPLICATIONS: In an attempt to answer to the growing demand in the fresh-cut produce industry to control browning, heat treatment was investigated as interesting alternative to chemical preservation methods. Efficient control of enzymatic browning in fresh-cut Iceberg lettuce could be achieved by heat treatment at 50°C for 60 s. Experimental data are provided showing the effects of wounding and subsequent heat treatment on visual browning, the BP and the activity of PAL, PPO, and POD during refrigerated storage up to 10 days. Using this data, correlations were found for BP, PAL activity, and POD activity, but not for PPO. Although undesired side effects of heat treatment (e.g., tissue softening) cannot be excluded, the obtained information might be useful for further research, serving as a baseline for wound-induced effects on browning-related parameters in fresh-cut lettuce and possible mechanisms of action of inhibitory treatments., (© 2019 Wiley Periodicals, Inc.)

Enzymatic browning is a crucial reaction affecting the quality of fresh-cut fruit and vegetables. Purslane is an edible Chinese folk medicine with extensive distribution and containing a lot of polyphenols and alkaloids. However, little research on its' anti-browning effect on fresh-cut food was reported. In this study, the effectiveness of 0.05% (w/w) purslane aqueous extract treatment efficiently inhibited the activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia-lyase (PAL), the membrane integrity was effectively maintained, and malondialdehyde (MDA) content increases was retarded during whole storage period at 4 °C. Oddly, the higher purslane extract concentration, the lower endogenesis phenolic content. Additionally, thirty polyphenols and fifty-six alkaloids were found in purslane aqueous extract by LC-MS/MS. All results suggest that purslane aqueous extract is a promising nutritive anti-browning agent for fresh-cut potato., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato l-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of their binding performed using molecular modeling have shown that they might be bound either in active site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby the to the entrance channel for substrate into active site., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The mechanisms of extreme Al-resistance in Urochloa decumbens are not established. Full resistance expression requires a lag time of 72-96h and is preceded by a sensitive phase (24-48h) with Al-induced root growth inhibition. The aim here was to identify key processes of the activation phase of Al-resistance analysing both root exudates and comparative root proteome. Samples were taken after 0, 24 and 96h exposure to 0 or 200μM Al. Al-induced stimulation of citrate and oxalate efflux was limited to the sensitive phase. Only 11 proteins revealed Al-induced abundance differences; six were identified. After 24h, phenylalanine ammonium lyase (PAL), methionine synthase (MS), and deoxymugineic acid synthase (DMAS) decreased, while acid phosphatase (APase) abundance increased. Coincident with growth recovering, PAL and MS, but not DMAS, returned to initial levels. After 96h, γ‑carbonic anhydrase (γ‑CA) and adenylate kinase (AK) along with two unidentified proteins were more abundant. In conclusion, few protein changes characterize the initial response to Al in signalgrass. During the alarm phase, changes are related to P-mobilization, downregulation of Fe-acquisition, reduction of phenolic biosynthesis, and small stimulation of organic acid exudation. After recovering (resistant phase), biosynthesis of phenolics and methionine, but not Fe-mobilization are re-established. Full expression of Al-resistance is characterized by enhanced γ‑CA mediating mitochondrial complex I assembly and increased AK abundance indicating higher root respiration and better provision of ADP and Mg 2+ to ATP synthase, respectively. The unidentified proteins and the specific role of γ‑CA in Al resistance of U. decumbens will centre future research., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Downy mildew of pearl millet caused by the biotrophic oomycete Sclerospora graminicola is the most devastating disease which impairs pearl millet production causing huge yield and monetary losses. Chitosan nanoparticles (CNP) were synthesized from low molecular weight chitosan having higher degree of acetylation was evaluated for their efficacy against downy mildew disease of pearl millet caused by Sclerospora graminicola. Laboratory studies showed that CNP seed treatment significantly enhanced pearl millet seed germination percentage and seedling vigor compared to the control. Seed treatment with CNP induced systemic and durable resistance and showed significant downy mildew protection under greenhouse conditions in comparison to the untreated control. Seed treatment with CNP showed changes in gene expression profiles wherein expression of genes of phenylalanine ammonia lyase, peroxidase, polyphenoloxidase, catalase and superoxide dismutase were highly upregulated. CNP treatment resulted in earlier and higher expression of the pathogenesis related proteins PR1 and PR5. Downy mildew protective effect offered by CNP was found to be modulated by nitric oxide and treatment with CNP along with NO inhibitors cPTIO completely abolished the gene expression of defense enzymes and PR proteins. Further, comparative analysis of CNP with Chitosan revealed that the very small dosage of CNP performed at par with recommended dose of Chitosan for downy mildew management.

Five new geminal aminocycloalkanephosphonic acids (4 - 8) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia-lyase (PAL). Within the set of compounds which are related to 2-aminoindane-2-phosphonic acid (AIP, 3), a known powerful inhibitor of PAL, racemic 1-aminobenzocyclobutene-1-phosphonic acid (4), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine-derived anthocyanin synthesis in buckwheat., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

Fresh-cut lotus root slices were treated with 80nM 24-epibrassinolide (EBR) and then stored at 4°C for 8days to investigate the effects on cut surface browning. The results showed that EBR treatment reduced cut surface browning in lotus root slices and alleviated membrane lipid peroxidation as reflected by low malondialdehyde content and lipoxygenase activity. EBR treatment inhibited the activity of phenylalanine ammonia lyase and polyphenol oxidase, and subsequently decreased phenolics accumulation and soluble quniones formation. The treatment also stimulated the activity of peroxidase, catalase and ascorbate peroxidase and delayed the loss of ascorbic acid, which would help prevent membrane lipid peroxidation, as a consequence, reducing decompartmentation of enzymes and substrates causing enzymatic browning. These results indicate that EBR treatment is a promising attempt to control browning at cut surface of fresh-cut lotus root slices., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this study, the effects of electrolyzed oxidizing water (EOW) on the prevention of enzymatic browning of fresh-cut "Jiu Jinhuang" Chinese yam were investigated. The yams were immersed in the inhibitors for 25 min at 20 °C. Compared with the tap water (TW) treatment, the chromatic attributes were significantly different after 72 h of storage (P < 0.05). The activities of polyphenol oxidase (PPO, EC 1.10.3.1), peroxidase (POD, EC 1.11.1.7), and L -phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were inhibited when measured at 24 h. The contents of phenolic acids, including gallic and chlorogenic acid, in the group treated with the slightly acidic electrolyzed water (SAEW) were higher than those treated with TW and neutral electrolyzed water (NEW). The group treated with NEW had the highest total phenol content (P < 0.05, at 24 h), while the group treated with SAEW had the highest flavonoid content (P < 0.05) during storage. Without being treated with inhibitors, the Km and Vmax values of yam PPO were 0.0044 mol/L and 0.02627 U/min, respectively, and the Ki of samples treated with SAEW and citric acid (CA) were 15.6607 and 2.3969 μmol/L, respectively. These results indicate that EOW is beneficial as a browning inhibitor., (© 2015 Institute of Food Technologists®)

The short-day plant pharbitis (also called Japanese morning glory), Ipomoea nil (formerly Pharbitis nil), was induced to flower by poor-nutrition stress. This stress-induced flowering was inhibited by aminooxyacetic acid (AOA), which is a known inhibitor of phenylalanine ammonia-lyase (PAL) and the synthesis of indole-3-acetic acid (IAA) and 1-aminocycropropane-1-carboxylic acid (ACC) and thus regulates endogenous levels of salicylic acid (SA), IAA and polyamine (PA). Stress treatment increased PAL activity in cotyledons, and AOA suppressed this increase. The observed PAL activity and flowering response correlate positively, indicating that AOA functions as a PAL inhibitor. The inhibition of stress-induced flowering by AOA was also overcome by IAA. An antiauxin, 4-chlorophenoxy isobutyric acid, inhibited stress-induced flowering. Both SA and IAA promoted flowering induced by stress. PA also promoted flowering, and the effective PA was found to be putrescine (Put). These results suggest that all of the pathways leading to the synthesis of SA, IAA and Put are responsive to the flowering inhibition by AOA and that these endogenous factors may be involved in the regulation of stress-induced flowering. However, as none of them induced flowering under non-stress conditions, they may function cooperatively to promote flowering., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors., (© 2014 The American Society of Photobiology.)

The activation of programmed cell death (PCD) is often a result of complex signalling pathways whose relationship and intersection are not well understood. We recently described a PCD root hair assay and proposed that it could be used to rapidly screen genetic or pharmacological modulators of PCD. To further assess the applicability of the root hair assay for studying multiple signalling pathways leading to PCD activation we have investigated the crosstalk between salicylic acid, autophagy and apoptosis-like PCD (AL-PCD) in Arabidopsis thaliana. The root hair assay was used to determine rates of AL-PCD induced by a panel of cell death inducing treatments in wild type plants treated with chemical modulators of salicylic acid synthesis or autophagy, and in genetic lines defective in autophagy or salicylic acid signalling. The assay demonstrated that PCD induced by exogenous salicylic acid or fumonisin B1 displayed a requirement for salicylic acid signalling and was partially dependent on the salicylic acid signal transducer NPR1. Autophagy deficiency resulted in an increase in the rates of AL-PCD induced by salicylic acid and fumonisin B1, but not by gibberellic acid or abiotic stress. The phenylalanine ammonia lyase-dependent salicylic acid synthesis pathway contributed only to death induced by salicylic acid and fumonisin B1. 3-Methyladenine, which is commonly used as an inhibitor of autophagy, appeared to influence PCD induction in all treatments suggesting a possible secondary, non-autophagic, effect on a core component of the plant PCD pathway. The results suggest that salicylic acid signalling is negatively regulated by autophagy during salicylic acid and mycotoxin-induced AL-PCD. However, this crosstalk does not appear to be directly involved in PCD induced by gibberellic acid or abiotic stress. This study demonstrates that the root hair assay is an effective tool for relatively rapid investigation of complex signalling pathways leading to the activation of PCD.

Oxidative browning is a common and often severe problem in plant tissue culture systems caused by the accumulation and oxidation of phenolic compounds. The current study was conducted to investigate a novel preventative approach to address this problem by inhibiting the activity of the phenylalanine ammonia lyase enzyme (PAL), thereby reducing the biosynthesis of phenolic compounds. This was accomplished by incorporating 2-aminoindane-2-phosphonic acid (AIP), a competitive PAL inhibitor, into culture media of Artemisia annua as a model system. Addition of AIP into culture media resulted in significant reductions in visual tissue browning, a reduction in total phenol content, as well as absorbance and autoflourescence of tissue extracts. Reduced tissue browning was accompanied with a significant increase in growth on cytokinin based medium. Microscopic observations demonstrated that phenolic compounds accumulated in discrete cells and that these cells were more prevalent in brown tissue. These cells were highly plasmolyzed and often ruptured during examination, demonstrating a mechanism in which phenolics are released into media in this system. These data indicate that inhibiting phenylpropanoid biosynthesis with AIP is an effective approach to reduce tissue browning in A. annua. Additional experiments with Ulmus americana and Acer saccharum indicate this approach is effective in many species and it could have a wide application in systems where oxidative browning restricts the development of biotechnologies.

We report a novel regulatory mechanism by which reactive oxygen species (ROS) regulate fumonisin B1 (FB1)-induced cell death. We found that FB1 induction of light-dependent ROS production promoted the degradation of GFP-labeled chloroplast proteins and increased phenylalanine ammonia lyase (PAL) activity, PAL1 gene expression and SA content, while pretreatment with ROS manipulators reversed these trends. Moreover, treatment with H2O2 or 3-amino-1,2,4-triazole increased PAL activity, PAL1 gene expression and SA content. PAL inhibitor significantly blocked FB1-induced lesion formation and SA increase. Our results demonstrate that light-dependent ROS accumulation stimulates the degradation of chloroplastic proteins and up-regulates PAL-mediated SA synthesis, thus promoting FB1-induced light-dependent cell death., (Copyright © 2013. Published by Elsevier B.V.)

Background: Chemical suppression of arthropod herbivores is the most common approach to plant protection. Insecticides, however, can cause unintended, adverse consequences for non-target organisms. Previous studies focused on the effects of pesticides on target and non-target pests, predatory arthropods, and concomitant ecological disruptions. Little research, however, has focused on the direct effects of insecticides on plants. Here we demonstrate that applications of neonicotinoid insecticides, one of the most important insecticide classes worldwide, suppress expression of important plant defense genes, alter levels of phytohormones involved in plant defense, and decrease plant resistance to unsusceptible herbivores, spider mites Tetranychus urticae (Acari: Tetranychidae), in multiple, distantly related crop plants., Methodology/principal Findings: Using cotton (Gossypium hirsutum), corn (Zea mays) and tomato (Solanum lycopersicum) plants, we show that transcription of phenylalanine ammonia lyase, coenzyme A ligase, trypsin protease inhibitor and chitinase are suppressed and concentrations of the phytohormone OPDA and salicylic acid were altered by neonicotinoid insecticides. Consequently, the population growth of spider mites increased from 30% to over 100% on neonicotinoid-treated plants in the greenhouse and by nearly 200% in the field experiment., Conclusions/significance: Our findings are important because applications of neonicotinoid insecticides have been associated with outbreaks of spider mites in several unrelated plant species. More importantly, this is the first study to document insecticide-mediated disruption of plant defenses and link it to increased population growth of a non-target herbivore. This study adds to growing evidence that bioactive agrochemicals can have unanticipated ecological effects and suggests that the direct effects of insecticides on plant defenses should be considered when the ecological costs of insecticides are evaluated.

White campion (Silene latifolia) is a dioecious plant that emits 1,2-dimethoxybenzene (veratrole), a potent pollinator attractant to the nocturnal moth Hadena bicruris. Little is known about veratrole biosynthesis, although methylation of 2-methoxyphenol (guaiacol), another volatile emitted from white campion flowers, has been proposed. Here, we explore the biosynthetic route to veratrole. Feeding white campion flowers with [(13)C9]l-phenylalanine increased guaiacol and veratrole emission, and a significant portion of these volatile molecules contained the stable isotope. When white campion flowers were treated with the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid, guaiacol and veratrole levels were reduced by 50% and 63%, respectively. Feeding with benzoic acid (BA) or salicylic acid (SA) increased veratrole emission 2-fold, while [(2)H5]BA and [(2)H6]SA feeding indicated that the benzene ring of both guaiacol and veratrole is derived from BA via SA. We further report guaiacol O-methyltransferase (GOMT) activity in the flowers of white campion. The enzyme was purified to apparent homogeneity, and the peptide sequence matched that encoded by a recently identified complementary DNA (SlGOMT1) from a white campion flower expressed sequence tag database. Screening of a small population of North American white campion plants for floral volatile emission revealed that not all plants emitted veratrole or possessed GOMT activity, and SlGOMT1 expression was only observed in veratrole emitters. Collectively these data suggest that veratrole is derived by the methylation of guaiacol, which itself originates from phenylalanine via BA and SA, and therefore implies a novel branch point of the general phenylpropanoid pathway.

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division., Results: This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose., Conclusions: This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Chlorogenic acid (CGA), a product of the phenylpropanoid pathway, is one of the most widespread soluble phenolic compounds in the plant kingdom. Although CGA is known to have important roles in plant function, its relevance in plant de novo organogenesis is not yet understood. With a series of experiments, here we show that CGA has a potential role in shoot, root and root hair development. In the first phase of our investigation, we developed an efficient and novel thin cell layer (TCL) regeneration protocol for Hypericum perforatum which could bridge all the in vitro morphogenetic stages between single cell and complete plant. Tissues at different morphogenetic states were analysed for their phenolic profile which revealed that shoot differentiation from callus tissues of H. perforatum is accompanied by the onset of CGA production. Further, the relevance of CGA in de novo organogenesis was deciphered by culturing highly organogenic root explants on media augmented with various concentrations of CGA. Results of this experiment showed that CGA concentrations lower than 10.0 mg l⁻¹ did not affect shoot organogenesis, whereas, higher concentrations significantly reduced this process in a concentration-dependent manner. In spite of the differential concentration-dependent effects of CGA on shoot regeneration, supplementation of CGA did not have any effect on the production of lateral roots and root hairs. Interestingly, CGA showed a concentration-dependent positive correlation with lateral roots and root hairs production in the presence of α-naphthaleneacetic acid (NAA). When the culture medium was augmented with 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), induction of shoots, lateral roots and root hairs from the explants was significantly affected. Addition of an optimum concentration of CGA in these cultures partially restored all these organogenic processes., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

We examined basal defense responses and cytomolecular aspects of riboflavin-induced resistance (IR) in sugar beet-Rhizoctonia solani pathsystem by investigating H(2)O(2) burst, phenolics accumulation and analyzing the expression of phenylalanine ammonia-lyase (PAL) and peroxidase (cprx1) genes. Riboflavin was capable of priming plant defense responses via timely induction of H(2)O(2) production and phenolics accumulation. A correlation was found between induction of resistance by riboflavin and upregulation of PAL and cprx1 which are involved in phenylpropanoid signaling and phenolics metabolism. Application of peroxidase and PAL inhibitors suppressed not only basal resistance, but also riboflavin-IR of sugar beet to the pathogen. Treatment of the leaves with each inhibitor alone or together with riboflavin reduced phenolics accumulation which was correlated with higher level of disease progress. Together, these results demonstrate the indispensability of rapid H(2)O(2) accumulation, phenylpropanoid pathway and phenolics metabolism in basal defense and riboflavin-IR of sugar beet against R. solani., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Cinnamaldehyde treatment inhibited the browning of cut lettuce during cold storage. In this study, to clarify the mechanism of inhibitory action of cinnamaldehyde against the browning and to show its microbiological merit, its effect on the browning of cut lettuce was compared to that of mild heat treatment. Both cinnamaldehyde and mild heat treatments inhibited the induction of phenylalanine ammonia-lyase (PAL) activity because of cutting. As a result, the biosynthesis of polyphenols, which are substrates of polyphenol oxidase, was inhibited. This reduction of polyphenol synthesis caused the inhibition of the browning. Cinnamaldehyde treatment repressed the induction of PAL mRNA, while mild heat treatment did not repress its induction. The increase in microbes in cut lettuce treated with cinnamaldehyde was less than that treated with mild heat after 12 days.

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

The effect of phenylalanine ammonia-lyase (PAL) inhibition by 2-aminoindane-2-phosphonic acid (AIP) in copper-exposed diploid and tetraploid chamomile (Matricaria chamomilla ) roots has been studied in a short-term experiment (24 h). Cu evoked stronger induction of PAL activity and accumulation of soluble phenols, flavonols (quercetin and kaempferol), and lignin in diploid plants, whereas AlCl3-reactive flavonoids and phenolic acids did not differ with respect to ploidy. Amounts of hydrogen peroxide and superoxide also preferentially increased in diploid. Surprisingly, PAL activity was restored in both +AIP cultivars, being inversely correlated with the accumulation of free phenylalanine. Notwithstanding this, total soluble phenols and flavonols were more depleted in Cu+AIP diploid roots. Soluble proteins decreased in response to Cu, and AIP had no effect. Among free amino acids, proline increased more visibly in Cu+AIP diploid, suggesting that this could be a protective mechanism in conditions with depleted content of phenols. Decrease in potassium content was ploidy-independent, calcium increased in all Cu variants, and Fe increased in Cu-exposed tetraploid. Shoot Cu content did not differ in Cu-exposed cultivars, but diploid roots contained more Cu. AIP decreased root Cu but increased shoot Cu amounts in diploid, whereas tetraploid plants did not exhibit similar responses. These data indicate that inhibition of root phenolic metabolism by AIP was effective enough, allowing Cu to accumulate in diploid shoots. The present findings are discussed in the context of available data about AIP effects and with respect to the role of phenols in metal uptake.

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonia-lyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.

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

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 about 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.

Cell wall apposition (CWA) formation is one of the first lines of defence used by plants to halt invading fungi such as powdery mildew. Lignin is a complex polymer of hydroxylated and methoxylated phenylpropane units (monolignols) and lignification renders the cell wall more resistant to pathogen attack. The role of monolignol biosynthesis in CWA-mediated defence against powdery mildew penetration into cereals is demonstrated here using RNA interference (RNAi)-mediated gene silencing and enzyme-specific inhibitors. Thirteen cDNAs representing eight genes involved in monolignol biosynthesis were cloned from an expression sequence tag (EST) library derived from the epidermis of diploid wheat (Triticum monococcum) infected with Blumeria graminis f. sp. tritici (Bgt). Differential expression patterns were found for these genes in susceptible and resistant plants after infection. Transcripts of phenylalanine ammonia lyase (PAL), caffeic acid O-methyltransferase (CAOMT), ferulic acid hydroxylase (FAH), caffeoyl-CoA O-methyltransferase (CCoAMT), and cinnamyl alcohol dehydrogenase (CAD) were accumulated, particularly in the epidermis. RNAi-mediated transient gene silencing in the epidermis led to a higher penetration efficiency of Bgt than in the controls. Gene silencing also compromised penetration resistance to varying degrees with different genes against an inappropriate pathogen, B. graminis f. sp. hordei (Bgh). Co-silencing led to greater penetration of Bgt or Bgh than when the genes were silenced separately. Fluorescence emission spectra analyses revealed that gene silencing hampered host autofluorescence response at fungal contact sites. These results illustrate that monolignol biosynthesis is critically important for host defence against both appropriate and inappropriate pathogen invasion in wheat.

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K(M) = 1.75 +/- 0.44 mM and V(max) = 3.01 +/- 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K(in) = 3.38 +/- 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57 degrees C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.

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.

Previously it had been shown that calycosin and calycosin-7-O-beta-D-glucoside (CGs) accumulate in whole plants, mainly in leaves, of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus) plants in response to low temperature. In this work, it was demonstrated that the influences of different conditions on CGs biosynthesis, by examining the changes in CGs content, as well as the expression of related genes, including phenylalanine ammonia lyase (PAL1), cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), chalcone reductase (CHR), chalcone isomerase (CHI), isoflavone synthase (IFS), and isoflavone 3'-hydroxylase (I3'H). The seven gene mRNAs accumulated in leaves of A. mongholicus upon exposure to low temperature in a light-dependent manner, though they exhibited different expression patterns. Transcriptions of CHS, CHR, CHI, IFS, and I3'H of the calycosin-7-O-beta-D-glucoside pathway were all up-regulated when plants were transferred from 16 degrees C to 2 degrees C or 25 degrees C or from 2 degrees C (kept for 24 h) to 25 degrees C. However, fluctuations in temperature influenced differently the transcriptions of PAL1 and C4H of the general phenylpropanoid pathway in leaves. Moreover, the amount of PAL1 expression changed sharply up and down, consistent with the variation of the content of CGs. PAL enzyme activity appears to be the limiting factor in determining the CGs levels. The inhibitor of PAL enzyme, L-alpha-aminooxy-beta-phenylpropionic acid, almost entirely shut down CGs accumulation at low temperature. All these results confirmed that PAL1, as a smart gene switch, directly controls the accumulation of CGs in A. mongholicus plants, in a light-dependent manner, during low temperature treatment.

Six derivatives of 2-aminoindane-2-phosphonic acid and 1-aminobenzylphosphonic acid were synthesized. The compounds were tested both as inhibitors of buckwheat phenylalanine ammonia-lyase (in vitro) and as inhibitors of anthocyanin biosynthesis (in vivo). (+/-)-2-Amino-4-bromoindane-2-phosphonic acid was found to be the strongest inhibitor from investigated compounds. The results obtained are a basis for design of phenylalanine ammonia-lyase inhibitors useful in the enzyme structure studies in photo labelling experiments.

Hairy roots of Brugmansia candida produce the tropane alkaloids scopolamine and hyoscyamine. In an attempt to divert the carbon flux from competing pathways and thus enhance productivity, the polyamine biosynthesis inhibitors cyclohexylamine (CHA) and methylglyoxal-bis-guanylhydrazone (MGBG) and the phenylalanine-ammonia-lyase inhibitor cinnamic acid were used. CHA decreased the specific productivity of both alkaloids but increased significantly the release of scopolamine (approx 500%) when it was added in the mid-exponential phase. However, when CHA was added for only 48 h during the exponential phase, the specific productivity of both alkaloids increased (approx 200%), favoring scopolamine. Treatment with MGBG was detrimental to growth but promoted release into the medium of both alkaloids. However, when it was added for 48 h during the exponential phase, MGBG increased the specific productivity (approx 200%) and release (250- 1800%) of both alkaloids. Cinnamic acid alone also favored release but not specific productivity. When a combination of CHA or MGBG with cinnamic acid was used, the results obtained were approximately the same as with each polyamine biosynthesis inhibitor alone, although to a lesser extent. Regarding root morphology, CHA inhibited growth of primary roots and ramification. However, it had a positive effect on elongation of lateral roots.

Stored cut lettuce gradually turns brown on the cut section after several days of storage, because cutting induces phenylalanine ammonia-lyase (PAL) activity, the biosynthesis of polyphenol is promoted, and the polyphenols are oxidized by polyphenol oxidase. In this study, we screened for inhibitors of PAL derived from fermented broths of microbes and from foods and found that a cinnamon extract definitely inhibited PLA of cut lettuce. An active component was isolated by chromatographic procedures and was identified as trans-cinnamaldehyde. Browning of cut lettuce immersed in a solution containing trans-cinnamaldehyde was definitely repressed.

A series of 5-substituted derivatives of the potent phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindane-2-phosphonic acid (AIP; 2) were synthesized. The AIP analogues 3-7, with additional NO2, NH2, Me, Br, and OH groups, respectively, were tested as in vitro inhibitors of buckwheat PAL, and as in vivo inhibitors of anthocyanin biosynthesis. Within this series, the racemic 5-bromo (6) and 5-methyl (7) congeners were biologically most active (Table), although being ca. one order of magnitude less potent than AIP proper.

The committed biosynthetic reaction to benzoyl-coenzyme A in the marine bacterium "Streptomyces maritimus" is carried out by the novel prokaryotic phenylalanine ammonia lyase (PAL) EncP, which converts the primary amino acid L-phenylalanine to trans-cinnamic acid. Recombinant EncP is specific for L-phenylalanine and shares many biochemical features with eukaryotic PALs, which are substantially larger proteins by approximately 200 amino acid residues.

Interaction energies of phenylalanine ammonia-lyase (PAL) active site residues with a series of PAL inhibitors have been partitioned into electrostatic, exchange, delocalization, and correlation components and compared with analogous results obtained previously for leucine aminopeptidase (LAP). In the latter metalloenzyme, either of the two charged residues controls entirely relative inhibitor binding energies, while at least four residues are required to determine ligand relative stabilization in neutral PAL. Significant correlation with experimental inhibitory activity was found between the stabilization energy at gradually decreasing levels of theory (MP2, SCF) down to the first-order Heitler-London term. Contrary to the LAP case, where the electrostatic term was sufficient to reproduce experimentally observed trends, in the case of PAL, exchange repulsion effects also have to be considered. Computational protocol presented herein constitutes a promising way to incorporate the first principle calculation's accuracy into the process of rational binding affinity prediction, revealing the physical nature of the interactions, where successive approximations can be introduced in a systematic and justifiable manner.

Duckweed (Spirodela intermedia) was grown axenically on 1/2 strength Hutner's nutrient solution plus 1% sucrose, with the l-phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindan-2-phosphonic acid (AIP) at 0.0, 0.05, or 10 microM, at constant 25 degrees C and a light intensity of 300 micromol m(-2) s(-1) photosynthetically active radiation from CW fluorescent lamps. Growth with 10 microM AIP led to decreased frond area and fresh weight, but dry weight was unchanged. Microscopic examination of fronds revealed increased frond thickness and a lack of reticulate aerenchyma. Ultraviolet epifluorescence microscopy and UV-Vis spectroscopy of methanolic extracts confirmed the dose-dependent inhibition of secondary phenolic synthesis with the near total elimination of secondary phenolic accumulation at the 10 microM level. AIP-treated plants showed increased sensitivity to UV-B as shown by a reduced F(v)/F(m). The results provided direct evidence of the working hypothesis that phenols function to screen UV radiation from reaching photosynthetic tissues or damaging other sensitive tissues. A novel histochemical method employing zirconyl chloride to visualize phenols is discussed.

Salicylic acid (SA) is an important regulator of plant defense responses, and a variety of Arabidopsis mutants impaired in resistance against bacterial and fungal pathogens show defects in SA accumulation, perception, or signal transduction. Nevertheless, the role of SA-dependent defense responses against necrotrophic fungi is currently unclear. We determined the susceptibility of a set of previously identified Arabidopsis mutants impaired in defense responses to the necrotrophic fungal pathogen Botrytis cinerea. The rate of development of B. cinerea disease symptoms on primary infected leaves was affected by responses mediated by the genes EIN2, JAR1, EDS4, PAD2, and PAD3, but was largely independent of EDS5, SID2/ICS1, and PAD4. Furthermore, plants expressing a nahG transgene or treated with a phenylalanine ammonia lyase (PAL) inhibitor showed enhanced symptoms, suggesting that SA synthesized via PAL, and not via isochorismate synthase (ICS), mediates lesion development. In addition, the degree of lesion development did not correlate with defensin or PR1 expression, although it was partially dependent upon camalexin accumulation. Although npr1 mutant leaves were normally susceptible to B. cinerea infection, a double ein2 npr1 mutant was significantly more susceptible than ein2 plants, and exogenous application of SA decreased B. cinerea lesion size through an NPR1-dependent mechanism that could be mimicked by the cpr1 mutation. These data indicate that local resistance to B. cinerea requires ethylene-, jasmonate-, and SA-mediated signaling, that the SA affecting this resistance does not require ICS1 and is likely synthesized via PAL, and that camalexin limits lesion development.

Phenylalanine ammonia-lyase (PAL) catalyzes the beta-elimination of ammonia from L-phenylalanine to trans-cinnamic acid. A study of inhibition of PAL by phenol, ortho-cresol, and meta-cresol gave mixed inhibition; para-cresol is not an inhibitor. The calculated values of K(i) and alphaK(i) are phenol, K(i)=2.1+/-0.5 mM and alphaK(i)=3.45+/-0.95 mM; ortho-cresol, K(i)=0.8+/-0.2 mM and alphaK(i)=3.4+/-0.2 mM; meta-cresol, K(i)=2.85+/-0.15 mM and alphaK(i)=18.5+/-1.5 mM. The synergistic inhibition of the same inhibitors with glycine showed a lack of inhibition with the para-cresol/glycine pair, while mixed inhibition was observed with the ortho-cresol/glycine pair (K(i)=0.038+/-0.008 mM, alphaK(i)=0.13+/-0.04 mM) and phenol/glycine pair (K(i)=0.014+/-0.003 mM, alphaK(i)=0.058+/-0.01 M). The meta-cresol/glycine pair gave competitive inhibition (K(i)=0.36+/-0.076 mM). The strong synergistic inhibition observed implies that the inhibitors bind at the active site: in fact, the inhibitors used imitate the structure of the substrate. The order of synergistic inhibition is the same for the sites related to K(i) and alphaK(i). These results are in agreement with the inhibitors entering two active sites located in two different subunits.

The conformationally restricted phenylalanine analogue 2-aminoindan-2-phosphonic acid (AIP) inhibits phenylalanine ammonia-lyase (PAL) competitively in a time-dependent manner. This phenomenon was investigated in more detail with the heterologously expressed, highly purified homotetrameric PAL-1 isozyme from parsley. The kinetic analysis revealed that the enzyme-inhibitor complex is formed in a single "slow" step with an association rate of k(2)=2.6+/-0.04 10(4) M(-1) s(-1). The inhibition is reversible with a dissociation rate of k(-2)=1.8+/-0.04 10(-4) s(-1) and an equilibrium constant of K(i)=7+/-2 nM. The previously described PAL inhibitor (S)-2-aminooxy-3-phenylpropanoic acid [(S)-AOPP] was also found to be a slow-binding inhibitor of PAL-1. The carboxyl analogue of AIP, 2-aminoindan-2-carboxylic acid, served as a substrate of PAL-1 and was converted to indene-2-carboxylic acid.

One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of L-phenylalanine to trans-cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40-50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor alpha-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 microM but having no effect at 10 microM. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.

[reaction: see text] We describe a highly convergent and efficient synthesis of (-)-pinolidoxin, a potent modulator of plant pathogenesis, providing unambiguous determination of the relative and absolute stereostructure of this highly oxygenated fungal metabolite. Our unique strategy highlights the applications of novel silacyclic precursors for stereocontrolled polyol synthesis and features the finding of the reversible ring-closing metathesis.

Dextrorotatory 1-amino-3',4'-dichlorobenzylphosphonic acid was found to be a potent inhibitor of the plant enzyme phenylalanine ammonia-lyase both in vitro and in vivo from among the ring-substituted 1-aminobenzylphosphonic acids and other analogues of phenylglycine. A structure activity relationship analysis of the results obtained permits predictions on the geometry of the pocket of the enzyme and is a basis in the strategy of better inhibitor synthesis.

The influence of the allelochemicals ferulic (FA) and vanillic (VA) acids on peroxidase (POD, EC 1.11.1.7) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activities and their relationships with phenolic acid (PhAs) contents and root growth of soybean (Glycine max (L.) Merr.) were examined. Three-day-old seedlings were cultivated in nutrient solution containing FA or VA (0.1 to 1 mM) for 48 h. Both compounds (at 0.5 and 1 mM) decreased root length (RL), fresh weight (FW) and dry weight (DW) and increased PhAs contents. At 0.5 and 1 mM, FA increased soluble POD activity (18% and 47%, respectively) and cell wall (CW)-bound POD activity (61% and 34%), while VA increased soluble POD activity (33% and 17%) but did not affect CW-bound POD activity. At 1 mM, FA increased (82%) while VA reduced (32%) PAL activities. The results are discussed on the basis of the role of these compounds on phenylpropanoid metabolism and root growth and suggest that the effects caused on POD and PAL activities are some of the many mechanisms by which allelochemicals influence plant growth.

To understand the regulation of phenylalanine ammonia-lyase (PAL) activity in the corn smut fungus, Ustilago maydis, we examined the effects of different media, metabolic effectors (including aromatic amino acids), and environmental factors on induction and repression of PAL activity. PAL was detected only in cell extracts and not in the culture medium. U. maydis PAL is constitutively produced at a low level in all media tested but its regulation can be influenced by aromatic amino acids. L-Tryptophan (0.3 mM) induces PAL activity 3- to 5-fold but tryptophan analogs and tryptophan-related metabolites do not. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. No induction or inhibition of PAL activity was observed as a function of culture temperature, pH or light. PAL induction was repressed by glucose but not by its reaction product, t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be involved. This study shows that the regulation of U. maydis PAL is very different from the patterns known for plants and other fungi.

Cut lettuce stored at 4 degrees C gradually turned brown on the cut section after several days of storage. Three factors for enzymatic browning, the polyphenol content, polyphenol oxidase activity, and phenylalanine ammonia-lyase (PAL) activity, were examined during the cold storage of cut lettuce. A relationship between the browning and PAL activity was apparent. We tried to prevent this browning by using the two enzyme inhibitors, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the phenylpropanoid pathway, and glyphosate, an inhibitor of the shikimate pathway. AIP and glyphosate significantly inhibited the browning of cut lettuce. The polyphenol content and PAL activity were both reduced by the treatment with AIP. These results show that regulating the biosynthesis of polyphenols is essential to prevent the browning of cut lettuce.

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinnamic acid. Specific photolabeling of the enzyme was detected using biotinylated derivatives without the use of radioisotopes. The results indicated that the 4-[3-(trifluoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to elucidate regulation of phenylpropanoid biosynthesis.

Salicylates, the main phenolic glucosides of northern willow (Salix spp.), play an important role in plant-herbivore interactions. Salicylates are labile metabolites that are thought to undergo metabolic turnover. Salicylates are synthesized from phenylalanine (Phe) via the shikimate pathway. 2-Aminoindan-2-phosphonic acid (AIP), a strong inhibitor of Phe ammonia-lyase (EC 4.3.1.5), was used to block the biosynthesis of salicylates. The aim of this study was to investigate long-term turnover of salicylates in intact micropropagated plantlets of Salix myrsinifolia Salisb. The biosynthesis of salicylates was inhibited efficiently but not completely by 30 microM 2-aminoindan-2-phosphonic acid. Inhibitor treatment, aside from leading to a high accumulation of Phe, also led to an increase in tyrosine and tryptophan, indicating that 2-aminoindan-2-phosphonic acid may also inhibit enzymes other than Phe ammonia-lyase. Salicylates were shown to be unexpectedly stable metabolites that did not undergo marked metabolic turnover in intact plants; in leaves no significant turnover occurred, and in the stems the five salicylates studied were turned over slowly, with half-lives of 11 to 25 d. The total amount of salicylate in mature shoots decreased only 0.6% per day.

We earlier reported that when phenylalanine ammonia-lyase (PAL) activity in radish seedlings was inhibited by the competitive inhibitor 2-aminoindan-2-phosphonic acid (AIP), soluble sinapoyl esters carried over from the seed were converted to wall-bound esters in young cotyledons. We now report that these soluble sinapoyl esters may also be converted into lignin in the cotyledons. When radish seedlings were grown in the presence of 100 microM AIP, lignin formation (determined as lignothioglycolic acid) was inhibited ca. 74% in the cotyledons and ca. 80% in hypocotyls plus roots. The syringyl to guaiacyl (S/G) ratio in the lignin of AIP-grown plants, as determined by alkaline cupric oxidation and from Fourier-transform infrared (FT-IR) spectra, was higher in cotyledons, but lower in hypocotyls plus roots, as compared to plants grown on distilled water. These results support the view that soluble sinapoyl esters preformed in seeds may contribute to the syringyl moiety of lignin in cotyledons during early seedling development and that there is no appreciable transport of soluble sinapoyl esters from cotyledons to the hypocotyls and roots.

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.