Cloning, expression and characterization of phenylalanine ammonia-lyase from Rhodotorula glutinis

The industrial-scale production of phenylalanine ammonia-lyase (PAL) mainly uses strains of Rhodotorula. However, the PAL gene from Rhodotorula has not been cloned. Here, the full-length gene of PAL from Rhodotorula glutinis was isolated. It was 2,121 bp, encoding a polypeptide with 706 amino acids and a calculated MW of 75.5 kDa. Though R. glutinis is an anamorph of Rhodosporium toruloides, the amino acid sequences of PALs them are not the same (about 74 % identity). PAL was expressed in E. coli and characterized. Its specific activity was 4.2 U mg^?1 and the k _cat/K _m was 1.9 × 10⁴ mM^?1 s^?1, exhibiting the highest catalytic ability among the reported PALs. The genetic and biochemical information reported here should facilitate future application in industry.     

Differential expression of phenylalanine ammonia-lyase genes in barley induced by fungal infection or elicitors

Gene-specific probes were used to assess the expression patterns of four different phenylalanine ammonia-lyase ( pal ) genes in infected or elicitor-treated leaves and suspension-cultured cells of barley. Genes corresponding to hpal2 , hpal3 , hpal4 , and hpal6 were all induced by mercuric chloride and fungal infection by Bipolaris sorokiniana Sacc. (Shoem.) in barley ( Hordeum vulgare L. cv. Pokko) leaves, but with considerable variation in their expression level and timing. The expression patterns of hpal2 and hpal6 were similar, both showing a rapid, strong induction after treatment with mercuric chloride and a slower induction after fungal inoculation, whereas the more divergent hpal3 was induced at a later time and at a lower level after both treatments. Hpal4 was expressed with timing like that of hpal2 and hpal6 in infected or mercuric chloride-treated leaves but its expression was much weaker. Hpal2 and hpal4 were induced in elicitor-treated, suspension-cultured barley cells, whereas the expression of hpal3 was nearly undetectable, and hpal6 was strongly and constitutively present. All pal genes except hpal4 were developmentally regulated, but differentially expressed in various barley tissues. The results suggest that the four pal genes studied here might be responsible for the activation of different branches in the phenylpropanoid biosynthesis of barley.     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

An inactivator of phenylalanine ammonia-lyase from gherkin hypocotyls

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

Exon shuffling in anther-specific genes from sunflower

We describe here the nucleotide sequence of an anther-specific gene (sf18) from sunflower, encoding a proline- and glycine-rich polypeptide with a hydrophobic amino-terminus (signal peptide). The gene is split by a 211 by intron and is partially related to another anther-specific gene (sf2) from sunflower with which it shares important sequence stretches in the 5 coding and upstream regions. We propose that the two genes have originated via exon shuffling, during which a copy of a DNA segment including the promoter region as well as a signal peptide coding sequence has been transferred into the upstream region of two different potential coding sequences, generating two novel genes which display the same specificity of expression and which both encode an extracellular protein. While the 5 region of the intron is highly conserved as part of the transferred region and may play a role in the selection of the 5 splice site, a common octanucleotide at the 3 end of the intron of the two genes might be involved in 3 splice site selection.     

Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen

Lignins, along with condensed tannins (CTs) and salicylate-derived phenolic glycosides, constitute potentially large phenylpropanoid carbon sinks in tissues of quaking aspen (Populus tremuloides Michx.). Metabolic commitment to each of these sinks varies during development and adaptation, and depends on L-phenylalanine ammonia-lyase (PAL), an enzyme catalyzing the deamination of L-phenylalanine to initiate phenylpropanoid metabolism. In Populus spp., PAL is encoded by multiple genes whose expression has been associated with lignification in primary and secondary tissues. We now report cloning two differentially expressed PAL cDNAs that exhibit distinct spatial associations with CT and lignin biosynthesis in developing shoot and root tissues of aspen. PtPAL1 was expressed in certain CT-accumulating, non-lignifying cells of stems, leaves, and roots, and the pattern of PtPAL1 expression varied coordinately with that of CT accumulation along the primary to secondary growth transition in stems. PtPAL2 was expressed in heavily lignified structural cells of shoots, but was also expressed in non-lignifying cells of root tips. Evidence of a role for Pt4CL2, encoding 4-coumarate:coenzyme A ligase, in determining CT sink strength was gained from cellular co-expression analysis with PAL1 and CTs, and from experiments in which leaf wounding increased PAL1 and 4CL2 expression as well as the relative allocation of carbon to CT with respect to phenolic glycoside, the dominant phenolic sink in aspen leaves. Leaf wounding also increased PAL2 and lignin pathway gene expression, but to a smaller extent. The absence of PAL2 in most CT-accumulating cells provides in situ support for the idea that PAL isoforms function in specific metabolic milieus.     

The effect of herbicides,plant growth regulators and other compounds on phenylalanine ammonia-lyase activity

The in vitro and in vivo effects of a number of herbicides and plant growth regulators on phenylalanine ammonia-lyase (PAL) activity were investigated. The most elective in vitro inhibitors were product analogs, t-cinnamic and p-coumaric acids, and carbonyl reagents, hydroxylamine and nitromethane. Application of the herbicides diuron, dalapon, amiben, and chloropropham, to plants resulted in a decrease in the intracellular concn of PAL. The inhibitory effect of alachlor was found to be dose-responsive and somewhat specific. A correlation between PAL inhibition and herbicidal activity was observed for hydroxylamine. The cytokinin, pyranyl benzyladenine, (PBA) increased PAL activity in pigweed. The possibility of developing herbicides acting through PAL inhibition is discussed.     

The characterization of phenylalanine ammonia-lyase from several plant species

Inhibition of the enzyme phenylalanine ammonia-lyase is considered as a target for the design of herbicides. A reliable and simple assay for the enzyme has been used and the kinetics of the enzyme from several sources compared. Purification of the enzyme from the grass green foxtail (Setaria glauca) did not change its kinetic behavior. The distribution of phenylalanine ammonia-lyase and tyrosine ammonia-lyase activity in various plant species was determined.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.

Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway, and therefore plays a key role in both plant development and stress defense. In many plants, PAL is encoded by a multi-gene family, and each member is differentially regulated in response to environmental stimuli. In the present study, we report that PAL in cucumber (Cucumis sativus L.) is encoded for by a family of seven genes (designated as CsPAL1-7). All seven CsPALs are arranged in tandem in two duplication blocks, which are located on chromosomes 4 and 6, respectively. The cDNA and protein sequences of the CsPALs share an overall high identity to each other. Homology modeling reveals similarities in their protein structures, besides several slight differences, implying the different activities in conversion of phenylalanine. Phylogenic analysis places CsPAL1-7 in a separate cluster rather than clustering with other plant PALs. Analyses of expression profiles in different cucumber tissues or in response to various stress or plant hormone treatments indicate that CsPAL1-7 play redundant, but divergent roles in cucumber development and stress response. This is consistent with our finding that CsPALs possess overlapping but different cis-elements in their promoter regions. Finally, several duplication events are discussed to explain the evolution of the cucumber PAL genes.     

Phenylalanine-dependent de novo synthesis of phenylalanine ammonia-lyase from Rhodotorula glutinis

A synthetic medium was developed in which the presence of phenylalanine ammonialyase (PAL) in the yeast Rhodotorula glutinis was dependent on the addition of l-phenylalanine. The appearance of PAL activity occurred during mid- to late log phase regardless of the time of l-phenylalanine introduction into the medium. Maximum levels of PAL activity were followed by a rapid decline in both total and specific activity. These changes were accompanied by comparable fluctuations in PAL antigen levels as measured by rocket immunoelectrophoresis. Proteins of yeast grown in the presences of l-phenylalanine were radiolabeled in vivo with l-[³H]leucine. The labeled protein was immunoprecipitated with anti-PAL serum and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A labeled protein comigrated with subunits of authentic PAL. These data support the hypothesis that de novo synthesis of PAL in R. glutinis is l-phenylalanine dependent.     

Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.     

A diverse family of phenylalanine ammonia-lyase genes expressed in pine trees and cell cultures

Using degenerate PCR primers that target evolutionarily conserved sequences in pal genes, we show that in the gymnosperm, Pinus banksiana, phenylalanine ammonia-lyase (PAL) is encoded by a multigene family of at least eight to ten loci. Five classes of pal sequence were easily distinguished among 28 clones sequenced from the products of PCR amplification of haploid genomic DNA. The dominant sequence from each class was named, yielding pal1 to pal5 loci. These genes shared 68.8% to 94.0% nucleotide identity over the 366 bp region compared. All of pal1 to pal5 were expressed in cell suspension cultures treated with a fungal elicitor and all but pal3 were expressed in differentiating xylem tissue of a mature tree. Only pal1 was expressed in unelicited cell cultures. While these P. banksiana genes are quite divergent, they are still more similar to each other than to any angiosperm pal gene cloned to date. For its roles in development and defense, PAL production in P. banksiana is coordinated from a large, diverse multigene family. We discuss evidence suggesting that other pines have similar pal gene family structures.     

Expression in Escherichia coli of catalytically active phenylalanine ammonia-lyase from parsley

In parsley (Petroselinum crispum), phenylalanine ammonia-lyase (PAL) is encoded by 4 structurally similar genes. The nucleotide sequence of a near full-length cDNA and the deduced amino acid sequence of PAL-4 are presented and compared with the corresponding sequences of PAL-1, a previously described representative of the gene family. Transformation of Escherichia coli cells with PAL-1 or PAL-4 cDNA yielded catalytically active PAL, suggesting that the catalytic center of the enzyme is formed spontaneously rather than by a plant-specific mechanism.