Gene cloning and enzymatic activity analysis of phenylalanine ammonia-lyase from Sinopodophyllum hexandrum (Royle) Ying / 生物工程学报

Phenylalanine ammonia-lyase (PAL) is the key entry enzyme of plant phenylpropanoid pathway. It plays an important role in the biosynthesis of podophyllotoxin, an anti-tumor lignan that is currently produced from its main natural source Sinopodophyllum hexandrum (Royle) Ying. In this study, we cloned the gene ShPAL encoding phenylalanine ammonia-lyase by RT-PCR from the root of S. hexandrum ecotype inhabited in the Aba' district, Sichuan, based on its public SRA transcriptome data-package. Bioinformatics analyses showed that the ShPAL-encoded protein is composed of 711 amino acids, contains the conserved domains of PAL, and has the signature motif within the active center of aromatic ammonia-lyases. Moreover, ShPAL protein was predicted to have a secondary structure mainly composed of α-helix and random coil, a typical 'seahorse' shape monomer tertiary structure, and a homologous tetramer three-dimensional structure by Swiss-Modelling. The phylogenetic lineage analysis indicated ShPAL was of the highest sequence identity and the shortest evolutionary distance with the PAL of Epimedium sagittatum from the same Berberidaceae family. Subcellular localization experiments showed that ShPAL protein was mainly distributed in the cytoplasm, despite of a minority on the endoplasmic reticulum membrane. Furthermore, ShPAL protein was recombinantly expressed in Escherichia coli and purified by histidine-tag affinity chromatography. Its enzymatic activity was determined up to 20.91 U/mg, with the optimum temperature of 41 ℃ and pH of 9.0. In contrast, the enzyme activity of its F130H mutant decreased by about 23.6%, yet with the same trends of change with temperature and pH, confirming that phenylalanine at this position does affect the substrate specificity of PAL. Both the wild type and the mutant have relatively poor thermostability, but good pH-stability. These results may help to further investigate the regulatory role of PAL in the process of podophyllotoxin biosynthesis and advance the heterologous synthesis of podophyllotoxin to protect the germplasm resource of S. hexandrum. They also demonstrate that ShPAL has a potential application in biochemical industry and biomedicine.

Semi-rational design improves the catalytic activity of phenylalanine ammonia lyase from Anabaena variabilis / 药学学报

Phenylalanine ammonia lyase (PAL) can catalyze L-phenylalanine to produce trans-cinnamic acid, which is widely used in the fields of pharmacy, food and agriculture. In particular, phenylalanine ammonia lyase from Anabaena variabilis (AvPAL) is the only protein drug for the treatment of phenylketonuria. However, the poor activity and low stability limit the application in industry of AvPAL. In this study, the key amino acids of substrate-binding cavity in AvPAL were identified by screening the single site saturation mutagenesis library. Subsequently, the impact of replacing M222 with the additional 19 amino acids on activity was also evaluated by site-directed mutagenesis. It was found that the kcat values of mutants M222L and M222V were 90% and 60% higher than that of AvPAL, and the kcat/Km was 1.4 and 1.5 times as that of AvPAL. Molecular docking results revealed that the higher activity of M222L and M222V may be due to the increase of hydrophobicity favorable for the substrate-binding cavity. This study is important for elucidating the structure-function relationship of AvPAL.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis / 中国天然药物

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

Cloning and expression analysis of phenylalanine ammonia-lyase gene in Lonicera macranthoides / 中草药

Objective To clone the full length of LmPAL1 gene and analyze bioinformatics and expression patterns from Lonicera macranthoides. Methods The total RNA of L. macranthoides was extracted. The full-length cDNA sequence of LmPAL1 gene was cloned by RT-PCR and RACE technique; The genome sequence in bioinformatics was analyzed by using the relevant software; The relative expression of the gene in stem, leaf, and different flower period was determined by using real-time PCR. Results The cloned LmPAL1 gene open reading frame (ORF) was 2 145 bp, encoding 714 amino acids. It was predicted by bioinformatics analysis as hydrophilic protein, being located in the chloroplasts, containing PAL shielding structure domain (527-641 aa). This gene contained PAL/HAL active center sequence GTITASGDLVPLSYIAG (196-212 aa), which was highly similar to other phenylalanine ammonia-lyase. Real-time PCR results showed that the relative expression level of golden yellow flowering flower was higher in seven florescence periods. When comparing the stem, leaf, and white flower bud period, the relative expression of flower was the highest and the leaf was the lowest. Conclusion In this study, PAL1 gene of L. macranthoides was cloned successfully, laying a foundation for further study of the function of this gene and genetic improvement of L. macranthoides quality and providing the research basis for exploring the biosynthesis and regulation of chlorogenic acid in L. macranthoides.

Cloning, sequence characterization, expression profiling of hormones response of phenylalanine ammonia-lyase gene in Bletilla striata / 中草药

Objective: Phenylalanine ammonia-lyase gene (PAL) is one of the key enzymes associated with stress resistance in secondary metabolism pathway of plants. Exploring its sequence information and expression profiling information in stress response could comprehensively peep at the protein structure, functions and signal network of plant stress resistance. Methods: The full cDNA length of PAL from Bletilla striata was cloned by RT-PCR and RACE approaches. Physicochemical properties and conserved domain of BsPAL protein were determined by a series of bioinformatics tools as Protparam, SOPMA, SWISS-MODEL, etc. Multiple alignment and phylogenetic tree were achieved by DNAMAN and MEGA Software, respectively. The qPCR was employed to examine the expression profiles of BsPAL under exogenous hormone stress. Results: The full cDNA of BsPAL was 2 708 bp, encoding a 797 amino-acid protein with a molecular weight of 86 216.94 and an isoelectric point (pI) of 6.24. The BsPAL protein included the typical structural domain and active site of PALs in other plants, and without transmembrane region, which was more homologous with PALs of Dendrobium officinale and Phalaenopsis aphrodita. The qPCR Results: revealed the expression level of BsPAL in roots was much higher than that in leaves and stems. Under MeJA treatment, the expression trend of BsPAL was first gradually ascending and then descending, while SA treatment had the reverse effect. Conclusion: The BsPAL’s sequence characterizing, expression profiling and responding patterns against SA and MEJA provided a research basis for elucidating the metabolic pathways of phenylpropanoid and hormone signaling research in B. striata.

Cloning and expression of three PAL genes in Astragalus membranaceus / 中草药

Objective To disclose the molecular mechanism of calycosin-7-O-β-D-glucoside (CG) accumulation in Astragalus membranaceus, we cloned PAL genes and analyzed the expression patterns of them and changes of CG contents in different tissues of A. membranaceus. Methods PAL genes were cloned with the methods of homology cloning and RACE technique using the total RNA as template and the analysis of bioinformatics on the cloned genes was carried out, gene expressions in root, stem, and leaf were determined with real-time PCR method, and CG content in root, stem, and leaf were analyzed by HPLC methods. Results Three PAL genes were cloned from A. membranaceus. The genbank accession number was KY086279 (AmPAL1), KY086280 (AmPAL2), and KY086281 (AmPAL3), respectively; The full-length cDNA of them was 2 508 bp, 2 401 bp, and 2 498 bp, respectively; And they all consisted of 2 157 bp open reading frame encoding 718 amino acids. Deduced AmPAL proteins had typical active sequences of PAL proteins, they were homology with other PAL proteins, and they shared the highest identities with PAL proteins of leguminous plants. Phylogenetic tree analysis showed AmPAL1 belonged to the different sub-class with the sub-class of AmPAL2 and AmPAL3. Real-time PCR analysis indicated that expression levels of AmPALs were different from each other, the expression level of AmPAL1 was the highest, the expression level of AmPAL2 was the next, and that of AmPAL3 was lowest in all detected tissues, and only the expression levels of AmPAL2 was similar to the changes of CG contents in different tissues (root > stem > leaf). Conclusion The cloned AmPAL1, AmPAL2, and AmPAL3 from A. membranaceus were typical genes of PAL, each might have different function in developing of different tissues, and AmPAL2 might involve in CG accumulation in different tissues.

Directing construction of CRISPR/Cas9 vector of SmPAL1 in Salvia miltiorrhiza by target efficiency detection in vitro / 中国中药杂志

To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences (SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.

New advances in phenylketonuria therapy research / 国际儿科学杂志

Phenylketonuria is a most common group of genetic metabolic diseases.Phenylketonuria is caused by enzymatic defects in the metabolic pathway,which is characterized by high blood phenylalanine concentration.Patients need early,reasonable treatment once diagnosis,otherwise there will be serious nervous system sequelae.Available treatments aim to decrease the blood phenylalanine concentration,reduce nervous system symptoms.The current primary treatment of phenylketonuria is the limitation of dietary phenylalanine intake.Considering the poor compliance with long-term eating restrictions and the heavy family burden,the application of new medicine such as trahydropterina cofactor,glycomacropeptide,large neutral amino acids can improve the therapeutic effect and living condition of phenylketonuria patients.In addition,recombinant phenylalanine ammonia lyase,hepatocyte transplantation,gene therapy,probiotics and other new treatments also seem to be a promising approach in the near future.

Induction of defense-related enzymes in patchouli inoculated with virulent Ralstonia solanacearum

Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis / 中国天然药物

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.

Potencial de preparados de cavalinha (Equisetum sp. ) na síntese de metabólitos de defesa em cotilédones de soja (Glycine max L. ) e o efeito sobre o crescimento de Rhizoctonia solani Kuhn, in vitro / Potential of horsetail (Equisetum sp. ) derivatives on the synthesis of defense metabolites using soybean ( Glycine max L. ) cotyledons and their effect on the in vitro growth of Rhizoctonia solani Kuhn

Foram desenvolvidos dois experimentos com objetivo de avaliar o potencial de preparados de cavalinha (Equisetum sp.) na síntese de metabólitos de defesa em cotilédones de soja (Glycinemax L.) e o efeito sobre o crescimento de Rhizoctonia solani, in vitro. O delineamento experimental utilizado para os experimentos foi inteiramente casualizado em esquema fatorial 3x5 (formas de extração x concentrações), com quatro repetições. As formas de extração foram extrato alcoólico, infusão e maceração, nas concentrações de zero; 1; 10, 20 e 40%. No primeiro experimento foi avaliada a indução de compostos de defesa vegetal em cotilédones de soja em resposta aos derivados a base de cavalinha, sendo quantificada a atividade da enzima fenilalanina amônia-liase (FAL), via espectofotometria, a fitoalexina gliceolina, e o teor de fenóis totais. No segundo experimento, in vitro, a unidade experimental foi uma placa de Petri, sendo os preparados de cavalinha incorporados ao meio BDA (Batata-dextrose e Agar) e avaliado o crescimento micelial de R. Solani. Os preparados de extrato alcoólico, infusão e maceração de cavalinha apresentaram capacidade de indução das fitoalexinas gliceolinas em cotilédones de soja, bem como, ativaram o metabolismo de compostos fenólicos. Entre os preparados, o extrato alcoólico e a maceração, se sobressaem sobre a infusão. Os preparados de extrato alcoólico, infusão e maceração de cavalinha em todas as suas concentrações inibem o crescimento do fungo R. solani, in vitro. .

Two experiments were carried out in the Federal Technological University of Paraná - Dois Vizinhos Campus - with the aim to evaluate the potential of horsetail (Equisetum sp.) derivatives for the synthesis of defense metabolites in soybean (Glycine max L.) cotyledons and their effect on the in vitro growth of Rhizoctonia solani. The experimental design was completely randomized in a 3 x 5 factorial design (extraction form x concentration), with four replications. The extraction forms were alcoholic extract, infusion and maceration and the concentrations tested were zero, 1, 10, 20 and 40%. In the first experiment, we evaluated the induction of plant defense in soybean cotyledons as a response to horsetail derivatives through spectrophotometry according to phytoalexin glyceollin, phenylalanine ammonia lyase enzyme activity (PAL) and total phenols. In the second experiment, in vitro, the experimental unit was a Petri dish, and the horsetail derivatives were incorporated into medium culture (potato dextrose agar), and we evaluated the mycelial growth of R. solani. The alcoholic extract, infusion and maceration of horsetail derivatives presented phytoalexin glyceolin induction in soybean cotyledons, in addition to activating the metabolism of phenolic compounds. Among the derivatives, the alcoholic extract and the maceration form of extraction were superior in relation to the infusion. The alcoholic extract, infusion and maceration of horsetail derivatives inhibited the in vitro growth of R. solani in all concentrations.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Effects of oligosaccharides from endophytic Fusarium oxysporum Dzf17 on activities of defense-related enzymes in Dioscorea zingiberensis suspension cell and seedling cultures

Background Three oligosaccharides (EOS, WOS and SOS) were respectively prepared from the corresponding polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharides (SPS) from the endophytic fungus Fusarium oxysporum Dzf17. In this study, the effects of EOS, WOS and SOS on the activities of the defense-related enzymes, namely phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD) in its host plant Dioscorea zingiberensis cultures were investigated. Results For the suspension cell cultures of D. zingiberensis, the highest PAL activity was induced by 0.5 mg/mL of WOS at 48 h after treatment, which was 4.55-fold as that of control. Both PPO and POD activities were increased to the maximum values by 0.25 mg/mL of WOS at 48 h after treatment, which were respectively 3.74 and 3.45-fold as those of control. For the seedling cultures, the highest PAL activity was elicited by 2.5 mg/mL of EOS at 48 h after treatment, which was 3.62-fold as that of control. Both PPO and POD reached their maximum values treated with 2.5 mg/mL of WOS at 48 h after treatment, which were 4.61 and 4.19-fold as those of control, separately. Conclusions Both EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.

cDNA cloning, Phylogenic Analysis and Gene Expression Pattern of Phenylalanine ammonia-lyase in Sugarcane (Saccharum officinarum L.)

The aim of the present study was to clone and characterize a full length cDNA of sugarcane (Saccharum officinarum) phenylalanine ammonia-lyase (SoPAL). Differential tissue expression pattern of the SoPAL transcript and its enzyme activity was also analyzed during the tillering stage of growth. The full-length of SoPAL cDNA was 2118 bp long and contained a protein with 706 amino acids, determined by encoding technique. The amino acid sequence and phylogenic analysis of the cloned SoPAL showed high similarity to PAL from other monocotyledonous such as sorghum (96%), maize (93%) and Bamboos (87.12%). The highest levels of SoPAL transcript were observed in the root and stem, while its minimal gene expression levels were in the leaves and sheath, respectively. The highest level of SoPAL enzyme activity was in the leaves. These results helped to understanding the characteristics of PAL biosynthesis and its regulation at the molecular level in sugarcane. This information could be critical for the manipulation of phenylpropanoid biosynthesis in the plant using biotechnological processes.

Phenylpropanoid enzymes, phenolic polymers and metabolites as chemical defenses to infection of Pratylenchus coffeae in roots of resistant and susceptible bananas (Musa spp.).

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.

Cloning and sequence analysis of phenylalanine ammonia-lyase gene from Curcuma longa / 中草药

Objective: This study aimed at cloning the phenylalanine ammonia-lyase (PAL) gene from Curcuma longa (CurPAL) and analyzing the bioinformatics. Methods: PAL gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from C. longa leaves. The bioinformatic analysis of this gene and its corresponding protein were performed. Results: One unique sequence of PAL, named as CurPAL (GenBank NO. KJ780359), was cloned from C. longa. The full-length of CurPAL cDNA was 1 293 bp, including 243 bp of 5'-UTR, 123 bp of 3'-UTR, and 927 bp of ORF encoding 308 amino acids. The molecular weight and theroretical isoelectric point (pI) of the deduced CurPAL protein were 33 000 and 5.76, respectively. The protein of CurPAL was stable and soluble. The domination sites and catalytic active sites in PAL protein of Nerium oleander were also found in CurPAL. It was found that the amino acid sequence of CurPAL had more than 75% homology with PAL of Prunus salicina, Camellia chekiangoleosa, Capsicum chinense, and Musa acuminate via multiple alignments. It revealed that CurPAL had closer relationship with PALs from Zingiberales plants than from other plants by phylogenetic tree analysis. Secondary and tertiary structures indicate that CurPAL is a full α protein contained by homotetramer. Conclusion: The cDNA encoding PAL from C. longa is cloned and reported for the first time. This work provides a scientific basis for exploring the biosynthetic pathway of the medicinal ingredient and improving its quality in C. longa.

Signal transduction of atractylodin biosynthesis in Atractylodes lancea cell induced by endophytic fungal elicitor mediated with nitric oxide followed by salicylic acid / 中草药

Objective: To investigate the signal molecules and signal transduction involved in endophytic fungal elicitor-induced atractylodin biosynthesis and the effect of an endophytic fungal elicitor on the key enzyme activity in Atractylodes lancea. Methods: Content changes of nitric oxide (NO), salicylic acid (SA), and atractylodin were detected under the endophytic fungal elicitor treatment by plant cell suspension culture technology. Results: The endophytic fungal elicitor remarkably promoted NO burst and the biosynthesis of SA and atractyodin by activating nitric oxide synthase (NOS), phenylalanine ammonia lyase (PAL), and acetyl coenzyme A carboxylase (ACC), respectively. NOS inhibitor PBITU could inhibit the NO and SA accumulation and the atractyodin biosynthesis induced by the elicitor. And atractyodin biosynthesis could also be triggered by exogenous NO or SA. The results indicated that NO and SA were the necessary signal molecules and NO burst was mediated by NOS induced by endophytic fungal elicitor. NO quencher cPITO could effectively remove NO burst in A. lancea cell induced by endophytic fungal elicitor and notably inhibit the biosynthesis promotion of SA and atractyodin in A. lancea cell induced by endophytic fungal elicitor. Exogenous SNP could reverse the cPITO inhibition on the activity of PAL and ACC and the synthesis of SA and atractylodin. This suggested that NO was an upstream signal molecule mediated endophytic fungal elicitor to accelerate the biosynthesis of SA and atractyodin. Conclusion: Endophytic fungal elicitor mediated through NO followed by SA could promote atractyodin biosynthesis by activating ACC in A. lancea.

Advances in research of molecular mechanism of phenylketonuria and its treatments / 国际生物医学工程杂志

Phenylketonuria (PKU) is a severe autosomal recessive disease which can cause irreversible damage to patients' neural system and results in severe mental retardation.Although the institution of a lowphenylalanine (Phe) diet has been a remarkable success in preventing the devastating damage associated with untreated PKU,there are always small but consistent gap in intelligence quotient (IQ) scores and executive functioning when compared to siblings or healthy age-related control groups.During the past few years,several types of new treatment strategies,such as genetic engineering,enzyme replacement,tetrahydrobiopterin (BH4),large neutral amino acids (LNAA),low-Phe diet and liver or liver cell transplantation therapies,have been studied and improved.This paper aims to introduce the research advances in pathogenesis of PKU,the treatment methods and the related molecular mechanism.

Activity of enzymes associated with the enzymatic browning of minimally processed potatoes

The purpose of the present study was to evaluate the effect of different potato cultivars and storage temperatures on the specific activity of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) in minimally processed potatoes. Potato cultivars Agata, Asterix and Monalisa were selected, washed, peeled, diced, sanitized, centrifuged, vacuum- packed and stored at 5 and 15°C for 9 and 5 days, respectively. There was an increase in the enzymatic activity in all the cultivars stored at 15°C. The cultivars 'Agata' and 'Asterix' stored at 5ºC did not differ significantly between them for the PAL, PPO and POD activities. The PAL, PPO and POD activities were also influenced by the storage temperature. The cultivars Agata and Asterix were more suitable in minimal processing than 'Monalisa', which was more susceptible to oxidative browning.

REGULACIÓN ESPACIO-TEMPORAL DE FENILALANLNA AMONIO LIASA EN CLAVEL (Dianthus caryophyllus L.) DURANTE SU INTERACCIÓN CON EL PATÓGENO Fusarium oxysporum f. sp. dianthi / SPATIO-TEMPORAL REGULATION OF PHENYLALANINE AMMONIA LYASE ENZYME IN CARNATION (Dianthus caryophyllus L.) DURING ITS INTERACTION WITH THE PATHOGEN Fusarium oxysporum f. sp. dianthi / REGULAÇÃO ESPÁCIO-TEMPORAL DA ENZIMA FENILALANINA AMÔNIO LIASA (PAL) EM CRAVO (Dianthus caryophyllus L.) DURANTE A SUA INTERACÇÃO COM O PATOGÉNICO Fusarium oxysporum f. sp. dianthi

Se estudió la regulación espacio-temporal de la actividad enzimática y de los niveles transcripcionales de la enzima fenilalanina amonio liasa (PAL por sus siglas en inglés) en clavel (Dianthus caryophyllus L.) inoculado con el patógeno causal del marchitamiento vascular. Se inocularon con Fusarium oxysporum f. sp. dianthi esquejes de dos variedades con diferencias en los niveles de tolerancia a la enfermedad -Kiss (tolerante) y Uconn (susceptible)- y se realizaron muestreos posinoculación a diferentes horas, tanto en el tallo como en la raíz, con el fin de evaluar los parámetros en estudio. Se determinó que durante la infección con el patógeno, la variedad tolerante presentó inducción de la actividad PAL en la raíz a las 48 horas de la inoculación. Esto indica que en este órgano de la planta se estimula la ruta fenilpropanoide, responsable de la generación de metabolitos fenólicos presentes en una alta diversidad de fenómenos asociados a resistencia vegetal. Considerando que durante la evaluación de los niveles de transcripción en este órgano de la planta mediante la técnica semicuantitativa de transcripción reversa y la posterior reacción en cadena de la polimerasa (RT-PCR por su nombre en inglés) no se presentó aumento importante en los niveles de mRNA para esta enzima, se evidencia la participación de mecanismos de regulación postranscripcional que determinan la generación de la enzima activa. En el tallo no se presentaron cambios en la actividad enzimática ni en los niveles transcripcionales, lo cual indica que la regulación de la respuesta de defensa de la planta está determinada por el órgano involucrado en el proceso de infección.

The spatio-temporal regulation of enzymatic activity and that of transcriptional levels of phenylalanine ammonia lyase enzyme in cuttings of carnation infected with the pathogen responsible of vascular wilting were studied. Carnation cuttings of two varieties which differ in disease tolerance-Kiss (tolerant) and Uconn (sus-ceptible)-were inoculated with Fusarium oxysporum f sp. dianthi and samplings were taken from root and stem at different times after inoculate them, to evaluate the parameters of interest. During infection, the tolerant variety showed an induction of PAL at 48 hours post-inoculation in roots, showing in this organ that the phenylpropanid pathway is stimulated. This pathway is responsible for producing phenolic metabolites that participate in phenomena associated with plant resistance. Having in account that during the evaluation of transcription levels in root, measured with semiquantitative technique RT-PCR, the levels of PAL mRNA were not increased, the participation of post-transcriptional regulation mechanisms for this active enzyme is evident. Taking in consideration that stem not presented differential induction in the enzymatic activity nor in levels of mRNA, we evidenced that regulation of responses of plant defense in this model is determined by the organ of the plant involved in the infection process.

com o fungo patogênico que causa o murchamente vascular. Inocularam-se estacas de cravo de duas variedades com diferentes niveles de tolerância à doença, Kiss (tolerante) e Uconn (susceptível) com uma Nesta investigação foi estudada a regulação espaço-temporal da atividade enzimática e dos níveis transcripcionais da enzima fenilalanina amônio liasa em estacas de cravo inoculadas cepa de Fusarium oxysporum f. sp. dianthi, e realizaram-se amostragens no caule e na raiz em diferentes horas pós-inoculação com a finalidade de avaliar os parâmetros em estudo. Foi determinado que durante a infecção com o fungo patogénico, a variedade tolerante Kiss apresenta uma indução da atividade PAL às 48 horas pós-inoculação na raiz, indicando que neste órgão da planta se estimula a via fenilpropanoide, responsável da geração de metabolitos de tipo fenólico que participam numa alta diversidade de fenómenos associados à resistência vegetal. Da mesma forma, considerando que durante a avaliação dos níveis de transcrição neste órgão da planta usando a técnica semi-quantitativa de RT-PCR não se apresentou aumento importante nos níveis de mRNA para esta enzima, se evidencia a participação de mecanismos de regulação postranscripcional que determinam a geração da enzima ativa. No caule não se apresentaram modificações na actividade enzimática, nem nos níveis de transcrição, indicando que a regulação da resposta de defesa da planta está determinada pelo órgão implicado no processo de infecção.