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. V_max, K_cat and K_m 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, Molecular ; Escherichia coli/metabolism* ; Phenylalanine/metabolism* ; Phenylalanine Ammonia-Lyase/chemistry* ; Recombinant Proteins ; Schisandra/genetics*

We studied the influence of the concentration of Ca2+ (0-50 mmol/L) in culture medium on the synthesis of rosmarinic acid (RA) and related enzymes in Salvia miltiorrhiza suspension cultures. Using verpamil (VP, a calcium channel antagonist) and ionophore A23187, we studied the mechanism of secondary metabolites of Salvia miltiorrhiza suspension cultures influenced by the concentration of Ca2+ in the culture medium. The synthesis of intracellular RA in 6-day incubation was significantly dependent on the medium Ca2+ concentration. At the optimal Ca2+ concentration of 10 mmol/L, a maximal RA content of 20.149 mg/g biomass dry weight was reached, which was about 37.3% and 20.4% higher than that at Ca2+ concentrations of 1 and 3 mmol/L, respectively. The variation of the activity of PAL and TAT, two key enzymes of the two branches of RA, could be affected by the concentration of Ca2+ in culture medium. The change of their activity occurred prior to the accumulation of RA, which suggested both of the key enzymes be involved in the synthesis of RA. Meanwhile, the enzymatic action of PAL was more distinct than TAT. The treatment of VP and A23187, respectively, indicated that the influence of RA affected by the concentration of Ca2+ in the culture medium was accomplished by the intracellular Ca2+, and the flow of Ca2+ from the extracellular to the intracellular environment could also participate in this process.
Calcium ; pharmacology ; Cinnamates ; metabolism ; Culture Media ; Culture Techniques ; methods ; Depsides ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Salvia miltiorrhiza ; chemistry ; enzymology ; growth & development ; Tyrosine Transaminase ; metabolism

We analyzed the best light intensity for callus induction and maintenance in Vitis vinifera and explored the mechanism of grape callus browning. Tender stem segments of grape cultivar "gold finger" were used to study the effects of different light intensities (0, 500, 1 000, 1 500, 2 000, 2 500, 3 000 and 4 000 Lx) on the induction rate, browning rate and associated enzyme activity and gene expression during Vitis vinifera callus formation. The callus induction rate under 0, 500, 1 000 and 1 500 Lx was more than 92%, significantly higher than in other treatments (P < 0.05). A lower browning rate and better callus growth were also observed during subculture under 1 000 and 1 500 Lx treatments. We found that chlorogenic acid, caffeic acid, p-hydroxybenzoic acid and coumaric acid contents were correlated with the browning rate of callus, among which chlorogenic acid content was positively correlated with the browning rate (P < 0.05). Peroxidase (POD) and polyphenol oxidase (PPO) activities were negatively correlated with the browning rate of callus (P < 0.01). The POD, PPO and phenylalanine ammonialyase (PAL) expression levels were positively correlated with the browning rate at P < 0.05 or P < 0.01. An appropriate light intensity for the tissue culture of Vitis vinifera was 1 000-1 500 Lx, higher or lower light intensities significantly impaired normal callus growth.
Caffeic Acids ; chemistry ; Catechol Oxidase ; chemistry ; Culture Media ; chemistry ; Gene Expression Regulation, Plant ; Light ; Peroxidase ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Stems ; enzymology ; radiation effects ; Tissue Culture Techniques ; Vitis ; enzymology ; radiation effects

OBJECTIVETo clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL.

METHODUsing homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods.

RESULTThe DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard.

CONCLUSIONThe PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fagopyrum ; classification ; genetics ; Molecular Sequence Data ; Phenylalanine Ammonia-Lyase ; chemistry ; genetics ; metabolism ; Phylogeny ; Protein Conformation ; Recombinant Proteins ; chemistry ; genetics ; metabolism

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.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Isatis ; enzymology ; genetics ; Molecular Sequence Data ; Multigene Family ; Phenylalanine Ammonia-Lyase ; chemistry ; genetics ; metabolism ; Phylogeny ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment

The increasing demand of Chinese materia medica could not be supplied by wild resource, and the cultivated medicinal materials become popular, which led to decreased quality of many medicinal materials due to the difference of the circumstance between the wild and the cultivated. How to improve quality becomes key points of Chinese medicine resource. The leaves of Scutellaria baicalensis were sprayed with H₂O₂, the activities of superoxide dismutase (SOD) and catalase (CAT) changed little, but there had been a marked decrease of peroxidase (POD) and ascorbic oxidase (APX), which showed that the antioxidase system declined. Meanwhile, H₂O₂, as enhanced the expression of phenylalnine ammonialyase (PAL) and β-glucuronidase (GUS) as well as activity of PAL, promoted the biosynthesis and biotransformation of flavonoids. At the day 2 after treated, H₂O₂ of 0.004 μmol·L⁻¹ the contents of the baicalin and the wogonoside decreased slightly, but the contents of the baicalein and the wogonin increased significantly, the baicalein from 0.094% to 0.324%, the wogonin from 0.060% to 0.110%, i. e. increased 246% and 83.3%, respectively.
Ascorbate Oxidase ; metabolism ; Catalase ; metabolism ; Drugs, Chinese Herbal ; chemistry ; Flavanones ; analysis ; Flavonoids ; analysis ; Glucosides ; analysis ; Glucuronidase ; metabolism ; Hydrogen Peroxide ; Peroxidase ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Scutellaria baicalensis ; metabolism ; Secondary Metabolism ; Superoxide Dismutase ; metabolism

The expression of phenylalnine ammonia lyase (LJPAL1) is closely related to the content of active compounds in Lonicera japonica. In this paper, a prokaryotic expression vector is constructed and LJPAL1 protein is expressed in E. coli. Three antigen sites were synthesized to peptide antigen and prepared polyclonal antibody of Anti-LJT-1, Anti-LJT-2 and Anti-LJT-3, separately. Antibody Anti-LJT-2 was screened using Western blotting. And indirect ELISA was built using Anti-LJT-2. The results of this study will be a base for honeysuckle chemical quality and evaluation kits.
Antibodies ; immunology ; metabolism ; Antibody Specificity ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Flowers ; chemistry ; Gene Expression Regulation, Plant ; Genetic Vectors ; Lonicera ; chemistry ; Phenylalanine Ammonia-Lyase ; chemistry ; immunology ; isolation & purification ; Plants, Medicinal ; chemistry ; Plasmids ; Protein Structure, Secondary ; Protein Structure, Tertiary

OBJECTIVEStudy on correlation between H2O2 scavenging system and flavonoids accumulation of Scutellaria baicalensis.

METHODThe content of baicalin and baicalein in suspension cell of S. baicalensis was determined by HPLC. The content of total flavonoids and H2O2, the activity of POD and PAL was detected by UV spectrophotometry.

RESULTThe content of total flavonoid and the activity of PAL increased significantly in 12 days after 40 degrees C, dark and PEG stress. Around 12 days after NPA, NPA +40 degrees C, 40 degrees C, NPA + dark, dark and PEG stress, the content of baicalin declined and the content of baicalein rise, the activity of POD showed an increasing trend, and level of H2O2 remain stable.

CONCLUSIONModerate environmental stress could promote the accumulation of total flavonoids in S. baicalensis, baicalin convert to baicalein by POD, and maintaining the stability of H2O2 content to avoid oxidative damage.

Anti-Infective Agents ; analysis ; metabolism ; Antioxidants ; analysis ; metabolism ; Cells, Cultured ; Darkness ; Flavanones ; analysis ; metabolism ; Flavonoids ; analysis ; metabolism ; Hot Temperature ; Hydrogen Peroxide ; analysis ; metabolism ; Oxidants ; metabolism ; Oxidative Stress ; Peroxidase ; metabolism ; Phenylalanine Ammonia-Lyase ; metabolism ; Plant Extracts ; chemistry ; Plant Roots ; chemistry ; drug effects ; enzymology ; metabolism ; Plants, Medicinal ; Polyethylene Glycols ; pharmacology ; Scutellaria baicalensis ; chemistry ; drug effects ; enzymology ; metabolism ; Stress, Physiological