Enzymes Of General Phenylpropanoid Metabolism Research Articles

Positive selection drives adaptive diversification of the 4-coumarate: CoA ligase (4CL) gene in angiosperms.

Lignin and flavonoids play a vital role in the adaption of plants to a terrestrial environment. 4-Coumarate: coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for both lignin and flavonoids biosynthesis. However, very little is known about how such essential enzymatic functions evolve and diversify. Here, we analyze 4CL sequence variation patterns in a phylogenetic framework to further identify the evolutionary forces that lead to functional divergence. The results reveal that lignin-biosynthetic 4CLs are under positive selection. The majority of the positively selected sites are located in the substrate-binding pocket and the catalytic center, indicating that nonsynonymous substitutions might contribute to the functional evolution of 4CLs for lignin biosynthesis. The evolution of 4CLs involved in flavonoid biosynthesis is constrained by purifying selection and maintains the ancestral role of the protein in response to biotic and abiotic factors. Overall, our results demonstrate that protein sequence evolution via positive selection is an important evolutionary force driving adaptive diversification in 4CL proteins in angiosperms. This diversification is associated with adaption to a terrestrial environment.

Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes

4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K m values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild-type K m values; enzymes with drastically altered K m values; and enzymes with almost wild-type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate-forming enzymes such as luciferases, peptide synthetases, and fatty acyl-CoA synthetases.

Extensive reprogramming of primary and secondary metabolism by fungal elicitor or infection in parsley cells.

The transcription rates of numerous plant genes have previously been shown to be strongly affected by pathogen infection or elicitor treatment. Here we estimate the extent and complexity of this response by analyzing the patterns of mRNA induction in fungal elicitor-treated parsley cells (Petroselinum crispum) for several representatives from various primary and secondary metabolic pathways, cytosolic as well as plastidic. As a reference, we use the biphasic accumulation curve for the coordinately induced mRNAs encoding the three core enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate:CoA ligase. Coincidence with this curve was observed for the mRNA induction kinetics of several, but not all, phenylpropanoid branch pathway-related reactions, whereas seven selected mRNAs from the pentose phosphate, glycolytic and shikimate pathways, including various cytosolic and plastidic isoforms, were induced with great differences in timing. Likewise unique and dissimilar from the reference curve were the induction patterns for various mRNAs encoding enzymes or proteins that are either more distantly or not at all related to phenylpropanoid metabolism. None of over 40 mRNAs tested so far remained unaffected. Using one strongly elicitor-responsive mRNA from carbohydrate metabolism, encoding a cytosolic glucose 6-phosphate dehydrogenase, for in situ RNA/RNA hybridization in fungus-infected parsley leaf tissue, we observed again the previously reported, close simulation of metabolic changes in true plant/fungus interactions by elicitor treatment of cultured cells. In addition to demonstrating extensive, highly complex functional, temporal and spatial patterns of changes in gene expression in infected plant cells, these results provide valuable information for the identification of pathogen-responsive promoters suitable for gene technology-assisted resistance breeding.

Distribution of Phenylalanine Ammonia Lyase and Chalcone Synthase within Trunks of Robinia pseudoacacia L.

Abstract:During heartwood formation, a kind of apoptosis in the inner parts of woody axes, phenolic substances are accumulated by in situ biosynthesis. In Robinia pseudoacacia L, these compounds are mainly flavonoids. In the present work, we performed a study to show if there is a correlation between measurable activities and detectable protein levels of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and chalcone synthase (CHS; EC 2.3.1.74), key enzymes of general phenylpropanoid metabolism and flavonoid biosynthesis, respectively. After separation of total protein extracts by one‐dimensional micro‐gel electrophoresis, newly emerging polypeptides were detectable within the sapwood‐heartwood transition zone, pointing toward a transient activation of metabolism shortly before cell death occurs. Most prominent was a polypeptide around 46 kDa. By immunoblotting, this band was identified as a CHS subunit. Thus, the exclusive presence of both enzyme protein and extractable enzyme activity of CHS in the heartwood bordering tissue was shown. In contrast, levels of PAL protein were similar in all xylem tissues which contain living cells. PAL activity, however, was measurable only in the differentiating xylem and the sapwood‐heartwood transition zone. From these results we conclude that during heartwood formation, CHS and PAL differ in their mode of regulation. It seems likely that CHS activity is regulated at the level of enzyme protein while PAL regulation is most probably post‐translational.

In situ Localization of Phenylpropanoid-Related Gene Expression in Different Tissues of Light- and Dark-Grown Parsley Seedlings

Using in situ RNA hybridization and immunohistochemical techniques, we investigated the tissue-specific and light-dependent expression of four genes involved in phenylpropanoid metabolism in various parts of parsley (Petroselinum crispum) seedlings. The genes encode phenylalanine ammonia-lyase (PAL) and 4-coum arate: CoA ligase (4CL), two enzymes of general phenylpropanoid metabolism; chalcone synthase (CHS), the key enzyme of flavonoid biosynthesis; and bergaptol O-methyltransferase (BMT), a late enzyme of the furanocoumarin pathway. In young leaves, PAL and 4 CL genes were expressed predominantly and in a coordinated fashion in epidermal cells, young xylem cells of vascular bundles, and epithelial cells of oil ducts. The gene(s) encoding BMT was active exclusively in the epithelial cells of oil ducts, whereas CHS gene expression was largely confined to the epidermis. In shoot and root apices, PAL, 4CL and CHS m RNAs were detectable at low levels without distinct patterns. The corresponding proteins, however, accumulated preferentially in the protoderm and pith meristem of the shoot apex and in root-cap cells. A gene encoding pathogenesis-related protein 1 (PR 1) was analyzed in roots for comparison and was found to be expressed predominantly in the cortical cells of root tips. The expression of all genes investigated was, to a greater or lesser extent, dependent on light conditions and tissue age, with highest levels occurring in newly differentiated, light-exposed tissue. In the leaf epidermis of seedlings illuminated for 20 h, PAL and CHS mRNAs and proteins, as well as 4CL protein, were particularly abundant.

Exonic sequences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression.

The parsley 4CL-1 gene encodes 4-coumarate:CoA ligase, a key enzyme of general phenylpropanoid metabolism. As well as being transcriptionally activated by such stresses as pathogen infection, UV-irradiation, and wounding, expression of 4CL-1 is developmentally regulated. In this paper we present evidence that 4CL-1 cis-acting elements which control stress-induced and developmental expression are physically separated. The ability of a series of 4CL gene constructions to respond to elicitor and light in stably or transiently transformed parsley cells was tested. While inducible expression was observed from all templates in which the 4CL-1 structural gene was fused to the 4CL-1 promoter, fusions of the promoter to the GUS reporter gene were completely unresponsive. The element(s) required for responsiveness appear to be exonic, since 4CL-1 introns and 3' flanking DNA had no effect on inducibility. Furthermore, this unconventional regulatory mode operates in transgenic tobacco plants, where we show that a 4CL-1 promoter fragment specifies correct cell-specific expression when fused to GUS yet is unresponsive to elicitor and light.

A parsley 4CL-1 promoter fragment specifies complex expression patterns in transgenic tobacco.

The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-beta-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in beta-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the distal portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.

Temporal and Spatial Patterns of Gene Expression around Sites of Attempted Fungal Infection in Parsley Leaves.

We analyzed the expression patterns of several pathogen defense-related genes in primary leaf buds of parsley by in situ RNA hybridization. Labeled antisense RNA probes were generated from seven selected cDNAs detecting transcripts from genes that are rapidly and strongly activated in cultured parsley cells upon treatment with fungal elicitor. These genes encode two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, a furanocoumarin-specific bergaptol O-methyltransferase, one pathogenesis-related protein, and three less well characterized proteins, designated as ELI 3, ELI 5, and ELI 7. In uninfected tissue, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNA levels were high in epidermal cells, oil-duct epithelial cells, and cells of the developing xylem; bergaptol O-methyltransferase mRNA was confined to oil-duct epithelial cells; and the pathogenesis-related protein and ELI 3, ELI 5, and ELI 7 mRNAs were undetectable. All seven mRNAs accumulated transiently and locally around infection sites caused by the soybean-pathogenic fungus Phytophthora megasperma f. sp. glycinea, to which parsley is nonhost-resistant. The observed late appearance of bergaptol O-methyltransferase mRNA, as compared with all other mRNAs, is in accord with a similar relative timing of transient gene activation in elicitor-treated cell cultures. Sharp borders were observed between the infection center, where hypersensitive cell death had occurred in response to fungal penetration, the surrounding area of local gene activation, and the remainder of the tissue not showing any apparent response. Some of the genes were also activated, although less sharply localized, upon wounding of parsley leaves.

Cellular localization of nonhost resistance reactions of parsley (Petroselinum crispum) to fungal infection

The response of parsley seedlings (Petroselinum crispum) inoculated with zoospores of the soybean-pathogenic fungus, Phytophthora megasperma f. sp. glycinea, ranged from "immunity" to "physiological susceptibility" depending on the post-inoculation environmental conditions. Typical nonhost resistance reactions, hypersensitive cell death and the formation of small local lesions, occurred under high relative humidity and 16 h illumination per day. Localized biochemical reactions were monitored using fluorescence microscopy combined with histochemical and immunohistochemical methods. The rapid accumulation of furanocoumarin phytoalexins, wall-bound phenolics and callose was detected around infection sites. Indirect antibody staining of frozen tissue sections demonstrated the local accumulation of phenylalanine ammonia-lyase, a key enzyme of general phenylpropanoid metabolism, and S-adenosyl-L-methionine: bergaptol O-methyltransferase, a specific enzyme of the furanocoumarin pathway. The results indicate that phenylpropanoid derivatives are synthesized de novo at infection sites.

Differential regulation and tissue-specific distribution of enzymes of phenylpropanoid pathways in developing parsley seedlings

Characteristic enzymes of general phenylpropanoid metabolism (phenylalanine ammonialyase) and of the flavonoid-glycoside and furanocoumarin branch pathways (chalcone synthase and S-adenosyl-L-methionine: bergaptol O-methyltransferase, respectively) were localized immuno-histochemically in cross-sections of various aerial parts of parsley (Petroselinum crispum) at different stages of seedling development. Phenylalanine ammonia-lyase occurred predominantly in epidermal and oil-duct epithelial cells, but was also detectable in other tissue parts. The two pathway-specific enzymes were localized in the epidermis (chalcone synthase) and in oil ducts (bergaptol O-methyl-transferase). High chalcone-synthase concentrations occurred very early in leaf development and then declined. High levels of the methyltransferase were present at all times investigated. The temporal and spatial at all times investigated. The temporal and spatial distribution of all three enzymes is in agreement with the time courses and sites of accumulation of the biosynthetic end products.

Occurrence of phytoalexins and other putative defense-related substances in uninfected parsley plants

Considerable amounts of the following substances were found in uninfected parsley (Petroselinum crispum) cotyledons: furanocoumarins, the putative phytoalexins of this and some related plant species, two enzymes of the furanocoumarin pathway (S-adenosyl-L-methionine: xanthotoxol and S-adenosyl-L-methionine: bergaptol O-methyltransferases), two hydrolytic enzymes (1,3-β-glucanase, EC 3.2.1.39, and chitinase, EC 3.2.1.14), and 'pathogenesis-related' proteins. The furanocoumarins and the methyltransferase activities reached their highest levels at the onset of cotyledon senescence as the hydrolytic enzymes increased from low to relatively high activity values. The relative amounts of pathogenesis-related proteins 1 and 2, as well as the corresponding mRNAs, also increased markedly. Two enzymes of general phenylpropanoid metabolism, L-phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, decreased in activity in a biphasic fashion during cotyledon development. At all developmental stages, the levels of these putative defense-related agents in total cotyledon extracts were too high to enable detection of, possibly, additional changes upon infection with zoospores of Phytophthora megasperma f. sp. glycinea, a fungal pathogen to which parsley shows a non-host, hypersensitive resistance response.

Structure and elicitor or u.v.-light-stimulated expression of two 4-coumarate:CoA ligase genes in parsley

We have isolated genomic clones encoding 4-coumarate:CoA ligase (4CL), a key enzyme of general phenylpropanoid metabolism, and have analysed the structure and regulation of the genes contained on these clones. Restriction enzyme and sequence analysis indicated that two distinct 4CL genes, Pc4CL-1 and Pc4CL-2, are represented on the clones and that additional 4CL genes are not present in parsley. Two lines of evidence suggest that each gene is transcriptionally activated by both elicitor and u.v. irradiation: cDNA clones corresponding to each gene were found in cDNA libraries made with RNA from both elicitor-treated and u.v-irradiated cells, and run-off transcripts homologous to a Pc4CL-2-specific intron probe were induced by both treatments. This induction was about half of the induction measured using probes homologous to both genes. The transcription initiation sites of both genes were determined. Comparison of the nucleotide sequences of the two genes 5' to these sites showed that they are highly homologous for several hundred base pairs and that they contain features potentially involved in regulation by elicitor and u.v. irradiation.

Responses of Cultured Parsley Cells to Elicitors from Phytopathogenic Fungi

Cultured parsley cells (Petroselinum crispum) responded to treatment with heat-released soluble cell-wall fragments (elicitors) from several different phytopathogenic fungi by forming coumarin derivatives (phytoalexins). This response was preceded in all cases by large but transient increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). The activities of two hydrolytic enzymes, chitinase and 1,3-beta-glucanase, also increased strongly in elicitor-treated cells, whereas the activities of three enzymes participating in primary metabolism were affected differently by the elicitor treatment. Glucose-6-phosphate dehydrogenase increased, phosphofructokinase remained almost constant, and pyrophosphate:fructose-6-phosphate phosphotransferase declined sharply in activity. Different amounts of cell-wall preparations from various phytopathogenic fungi were required for maximum elicitor activity. While three oomycetes (Phytophthora spp.) yielded the most active elicitors studied (maximum coumarin accumulation at concentrations of about 10 microgram per milliliter), cell-wall preparations from an ascomycete and three deuteromycetes gave comparable results only at 10 to 100 times higher concentrations. Optimal induction of PAL, 4CL, and chitinase with Phytophthora elicitor required only about 1 microgram per milliliter, whereas 1,3-beta-glucanase induction showed a dose dependence similar to that observed for coumarins. The elicitor concentration had pronounced effects not only on the extent, but also on the timing of all induced reactions.

Phytoalexin synthesis in soybean cells: Elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation

A glucan elicitor from cell walls of the fungus Phytophthara megasperma f. sp. glycinea, a pathogen of soybean ( Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspensioncultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.

Phytoalexin synthesis in soybean ( Glycine max) : Similar time courses of mRNA induction in hypocotyls infected with a fungal pathogen and in cell cultures treated with fungal elicitor

Rapid, transient increases in mRNA activities related to phytoalexin synthesis were observed after inoculation of soybean seedlings ( Glycine max) with the fungus, Phytophthora megasperma f. sp. glycinea, or treatment of cultured soybean cells with an elicitor preparation from the same fungus. The time courses of changes in activity were similar for mRNAs coding for enzymes of general phenylpropanoid metabolism and for chalcone synthase mRNA in both systems. Cloned cDNA was used to demonstrate that the induced changes in chalcone synthase mRNA activity coincided with changes in amount of mRNA. The results suggest that phytoalexin synthesis, one mechanism of induced resistance of plants to pathogens, is regulated by temporary gene activation.

Rapid Response of Suspension-cultured Parsley Cells to the Elicitor from Phytophthora megasperma var. sojae

Large and rapid increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, occurred in suspension-cultured parsley cells (Petroselinum hortense) treated with an elicitor preparation from Phytophthora megasperma var. sojae. Highest enzyme activities were obtained with an elicitor concentration similar to that required for maximal phenylalanine ammonialyase induction in cell suspension cultures of soybean, a natural host of the fungal pathogen.The changes in phenylalanine ammonia-lyase activity in parsley cells were caused by corresponding changes in the mRNA activity for this enzyme. Phenylalanine ammonia-lyase mRNA activity increased much faster and transiently reached a much higher level in elicitor-treated than in irradiated cell cultures. In contrast to irradiation, treatment of the cells with the elicitor did not induce the enzymes of the flavonoid glycoside pathway, as demonstrated for acetyl-CoA carboxylase and chalcone synthase. Induction of these enzymes by light was abolished by simultaneous application of the elicitor.

MRNA-dependent regulation of UDP-apiose synthase activity in irradiated plant cells.

Large changes in the rate of synthesis of UDP-apiose synthase, an enzyme of the flavonoid glycoside pathway, were observed both in vivo and in vitro following irradiation of previously dark-grown cell suspension cultures of parsley (Petroselinum hortense). Irradiation of the cells with ultraviolet light for 2 1/2 h caused a large increase in UDP-adipose synthase mRNA activity which continued for several hours during the subsequent dark period. A sharp peak in this activity was followed by a rapid exponential decline. The timing of changes in mRNA activity was very similar for UDP-apiose synthase and chalcone synthase, a previously investigated enzyme of the flavonoid glycoside pathway, but differed from that for phenylalanine ammonia-lyase, an enzyme of general phenylpropanoid metabolism. Thus, the present data provide further evidence for a differential regulation of the two groups of enzymes and a high degree of co-ordination within each group. The expected light-induced changes in UDP-apiose synthase activity were calculated from the observed changes in the rate of synthesis, allowing for an apparently constant rate of degradation (t 1/2 = 17 h). The resulting time course was in agreement with experimental data, suggesting that the changes in enzyme activity were caused by corresponding changes in the rate of synthesis. A protein of unknown function (Protein X), which is tightly associated with UDP-apiose synthase and co-purifies with the enzyme at a molar ratio of 1:1, is shown to be regulated independently of the enzymes of the flavonoid glycoside pathway.

Enzymes of General Phenylpropanoid Metabolism and of Flavonoid Glycoside Biosynthesis in Parsley

Several enzymes of phenylpropanoid metabolism showed large changes in their inducibility by light during the growth cycle of cell suspension cultures from parsley (Petroselinum hortense Hoffm.). Two of the three enzymes of general phenylpropanoid metabolism (group I) and six of the approximately 13 enzymes of the flavone and flavonol glycoside pathways (group II) were investigated. Both enzymes of group I (phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase) were most efficiently induced at two different stages: first, soon after starting a new culture, and second, near the beginning of the stationary phase. In contrast, the enzymes of group II (acetyl-coenzyme A carboxylase, flavanone synthase, chalcone isomerase, UDP-apiose synthase, and at least one of two malonyltransferases) were maximally induced during exponential growth of the culture. This result supports the conclusions drawn from previous data that the two groups are regulated differentially and that the enzymes within each group are regulated in a coordinated manner.