Evidence for p-hydroxybenzoate formation involving enzymatic phenylpropanoid side-chain cleavage in hairy roots of Daucus carota

In this study, methyl jasmonate (MJ)-elicited hairy root cultures of Daucus carota were explored to study the enzymatic route to p-hydroxybenzoic acid (p-HBA) biosynthesis. Treatment with 100muM MJ caused an enhanced accumulation of p-HBA as well as total phenolic content in elicited root lines as compared to untreated (controls) lines. Using cell-free extract as the source of crude enzymes, attempt was made to reveal the enzymatic route to p-HBA formation. The accumulation of p-HBA was preceded by a substantial upliftment of p-hydroxybenzaldehyde dehydrogenase (HBD) activity in elicited lines as compared to controls. A rapid 6-fold enhancement of phenylalanine ammonia-lyase (PAL) activity, the first enzyme of the phenylpropanoid pathway was also observed. Finally, we demonstrated here for the first time, in D. carota, the evidence of a quite unusual p-hydroxybenzaldehyde synthase (HBS)-type enzyme, which catalyzes the penultimate step of p-HBA biosynthesis by making phenylpropanoid side-chain cleavage of p-coumaric acid without involvement of any cofactor(s), but uplifted by supplementation of a thiol reagent such as DTT in the reaction buffer. This enzyme showed activity in a relatively broad pH range (7-8.4) and the temperature optimum was found to be at 34 degrees C. The MJ-treated roots showed highest HBS activity at 24h (52nkat/mg protein), which was nearly 5-fold higher than that in the control lines.     

Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development

The phenylpropanoid pathway yields a variety of phenolics that are closely associated with fruit qualities in addition to structural and defense-related functions. However, very little has been reported concerning its metabolism in fruit. This experiment was designed to assess changes of eleven phenolic acids in grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) and explore both the activities and amounts of three key enzymes--phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H) and 4-coumarate:coenzyme A ligase (4CL)--catalyzing the biosynthesis of these compounds during berry development. Finally, the subcellular localizations of the enzymes within berry tissues were also investigated using immuno-gold electron microscopic technique. The results indicated that the contents of gallic, protocatechuic, gentisic and caffeic acid all changed drastically during berry development, while other compounds containing p-hydroxybenzoic, vanillic, syringic, chlorogenic, p-coumaric, ferulic and sinapic acid varied only slightly. Activities of PAL, C4H and 4CL showed similar pattern changes with two accumulated peaks throughout berry development. In addition, their activities all showed a highly positive correlation with the total contents of phenolic acids, whereas the immunoblotting analysis showed that changes in enzyme activities were independent of the enzyme amounts. Results from the subcellular-localization study revealed that PAL was mainly present in the cell walls, secondarily thickened walls, and the parenchyma cells of the berry mesocarp cells, C4H was found primarily in the chloroplast (plastid) and nucleus and 4CL predominantly in the secondarily thickened walls and the parenchyma cells of mesocarp vascular tissue.     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway

3H-l-Phenylalanine is incorporated into a range of phenylpropanoid compounds when fed to tobacco cell cultures. A significant proportion of (3)H-trans-cinnamic acid formed from (3)H-l-phenylalanine did not equilibrate with exogenous trans-cinnamic acid and therefore may be rapidly channeled through the cinnamate 4-hydroxylase (C4H) reaction to 4-coumaric acid. Such compartmentalization of trans-cinnamic acid was not observed after elicitation or in cell cultures constitutively expressing a bean phenylalanine ammonia-lyase (PAL) transgene. Channeling between PAL and C4H was confirmed in vitro in isolated microsomes from tobacco stems or cell suspension cultures. This channeling was strongly reduced in microsomes from stems or cell cultures of transgenic PAL-overexpressing plants or after elicitation of wild-type cell cultures. Protein gel blot analysis showed that tobacco PAL1 and bean PAL were localized in both soluble and microsomal fractions, whereas tobacco PAL2 was found only in the soluble fraction. We propose that metabolic channeling of trans-cinnamic acid requires the close association of specific forms of PAL with C4H on microsomal membranes.     

Induction of defense responses in cultured parsley cells by plant cell wall fragments

Cell suspension cultures of parsley (Petroselinum crispum) accumulated coumarin phytoalexins and exhibited increased β-1,3-glucanase activity when treated with either a purified α-1,4-d-endopolygalacturonic acid lyase from Erwinia carotovora or oligogalacturonides solubilized from parsley cell walls by endopolygalacturonic acid lyase. Coumarin accumulation induced by the plant cell wall elicitor was preceded by increases in the activities of phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL) and S-adenosyl-l-methionine:xanthotoxol O-methyltransferase (XMT). The time courses for the changes in these three enzyme activities were similar to those observed in cell cultures treated with a fungal glucan elicitor. The plant cell wall elicitor was found to act synergistically with the fungal glucan elicitor in the induction of coumarin phytoalexins. As much as a 10-fold stimulation in coumarin accumulation above the calculated additive response was observed in cell cultures treated with combinations of plant and fungal elicitors. The synergistic effect was also observed for the induction of PAL, 4CL, and XMT activities. These results demonstrate that plant cell wall elicitors induce at least two distinct biochemical responses in parsley cells and further support the role of oligogalacturonides as important regulators of plant defense.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max)

The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H₂O₂) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H₂O₂ content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H + G + S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.     

Jasmonic acid elicitation of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones

Hypericum perforatum L. cell suspensions were evaluated for their viability, growth, dark gland formation and ability to produce phenylpropanoids and naphtodiantrones after elicitation with different jasmonic acid (JA) concentrations. Phenolic compounds were analyzed by high performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization mass spectrometry (ESI-MS). The activities of two key enzymes of the phenylpropanoid/flavonoid pathways, phenylalanine ammonia lyase (PAL) and chalcone isomerase (CHI) were also monitored to estimate general channeling in the different metabolic pathways. A 6-fold increase of phenolic compounds, flavanols and flavonols after JA elicitation was observed in cells. In contrast, anthocyanins were in lower amounts in JA treated cells suggesting a modification of the channeling in the phenylpropanoid pathway. Similar accumulations with maxima after 4 days of elicitation were found for naphtodianthrones (2.4-fold) such as hypericin and pseudohypericin in cells. At least a 6–8-fold increase of PAL and CHI activities was observed in JA elicited cells confirming a strong activation of the phenylpropanoid pathway. JA elicitation increased production of phenylpropanoids and naphtodianthrones in H. perforatum cell suspension without differentiation of dark glands under 16 h photoperiod.     

Influence of phosphate on the formation of the indole alkaloids and phenolic compounds in cell suspension cultures of Catharanthus roseus I. Comparison of enzyme activities and product accumulation

In cell suspension cultures of Catharanthus roseus a rapid accumulation of secondary compounds (tryptamine, indole alkaloids, phenolics) was observed after transfer of the cells into special ‘induction’-media devoid of phosphate and other essential growth factors [11, 14]. The increase of product levels was suppressed in the presence of phosphate which was almost completely taken up from the medium and accumulated by the cells within 48 h after inoculation. The activities of tryptophan decarboxylase (TDC), the first enzyme in indole alkaloid biosynthesis, and of phenyl-alanine ammonia-lyase (PAL), the key enzyme of phenylpropanoid biosynthesis, were influenced differently by phosphate. Whereas the accumulation of phenolics and PAL activity were similarly inhibited by low concentration of phosphate, the medium-induced enhanced activity of TDC was not affected although the product pools were considerably reduced. Some consequences for the regulation of secondary metabolism will be discussed.     

Phenylalanine ammonia-lyase activity and capsaicin-precursor compounds in p-fluorophenylalanine-resistant and -sensitive variant cells of chili pepper (Capsicum annuum)

Cell suspension cultures of chili pepper ( Capsicum annuum L. cv. Tampiqueño 74) displaying differences in their resistance to p -fluorophenylalanine (PFP) and in their contents of capsaicin (the compound which is responsible for the hot taste of chili pepper fruits) were characterized in relation to the activity of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the levels of free l -phenylalanine, phenolics and the phenylpropanoid acids involved in capsaicin biosynthesis. A nonselected cell line, a sensitive line (CA-02), a moderately resistant cell line (CA-29) and two resistant cell lines (CA-04 and CA-16) were studied. Higher PAL activities and higher levels of phenylalanine and phenolics were found in the PFP-resistant cells even after a minimum of 9 subcultures (15 days each) in the absence of the analog, indicating that the selected trait was stable. PFP-resistant chili pepper cells accumulated higher amounts of capsaicin precursors (cinnamic, caffeic and ferulic acids) than either the nonselected cells or the sensitive cell line. p -Coumaric acid was not detected at significant levels in any of the cell cultures. Overall, accumulation of free phenyl-alanine correlated well with PAL activity, phenolics, phenylpropanoids and capsaicin levels, suggesting an active flow through the phenylpropanoid pathway in PFP-resistant cells of chili pepper.     

Enzymes of phenylpropanoid metabolism in the important medicinal plant Melissa officinalis L.

Lemon balm (Melissa officinalis, Lamiaceae) is a well-known medicinal plant. Amongst the biologically active ingredients are a number of phenolic compounds, the most prominent of which is rosmarinic acid. To obtain better knowledge of the biosynthesis of these phenolic compounds, two enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL) and 4-coumarate:coenzyme A-ligase (4CL), were investigated in suspension cultures of lemon balm. MoPAL1 and Mo4CL1 cDNAs were cloned and heterologously expressed in Escherichia coli and the enzymes characterised. Expression analysis of both genes showed a correlation with the enzyme activities and rosmarinic acid content during a cultivation period of the suspension culture. Southern-blot analysis suggested the presence of most probably two gene copies in the M. officinalis genome of both PAL and 4CL. The genomic DNA sequences of MoPAL1 and Mo4CL1 were amplified and sequenced. MoPAL1 contains one phase 2 intron of 836 bp at a conserved site, whilst Mo4CL1 was devoid of introns.     

Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae

Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides, 4-coumarate:coenzyme A (CoA) ligase (4CL) from Arabidopsis thaliana, and chalcone synthase (CHS) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans-cinnamic acid and p-coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter(-1) of naringenin and 0.8 mg liter(-1) of pinocembrin. Several by-products, such as 2',4',6'-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l-tyrosine.     

Role of Jasmonates in the Elicitor- and Wound-Inducible Expression of Defense Genes in Parsley and Transgenic Tobacco

Jasmonates have been proposed to be signaling intermediates in the wound and/or elicitor-activated expression of plant defense genes. We used parsley (Petroselinum crispum) cell cultures and transgenic tobacco (Nicotiana tabacum) plants expressing 4CL1-GUS gene fusions to investigate the potential role played by jasmonates in mediating the wound and/or elicitor activation of phenylpropanoid and other defense-related genes. Jasmonates and [alpha]-linolenic acid strongly induced the expression of 4CL in a dose-dependent manner in parsley cells; methyl jasmonate also activated the coordinate expression of other phenylpropanoid genes and the accumulation of furanocoumarin phytoalexins. However, the response of the cells to optimal methyl jasmonate concentrations was distinct quantitatively and qualitatively from the response of elicitor-treated cells. In transgenic tobacco wound-inducible tobacco 4CL genes and a 4CL1 promoter-GUS transgene were responsive to jasmonates and [alpha]-linolenic acid in a dose-dependent manner. Pre-treatment of parsley cells or tobacco leaves with a lipoxygenase inhibitor reduced their responsiveness to the elicitor and to wounding. These results show that the elicitor response in parsley cells can be partially mimicked by jasmonate treatment, which supports a role for jasmonates in mediating wound-induced expression of 4CL and other phenylpropanoid genes.     

Induction of phenylpropanoid and tyramine metabolism in pectinase- or pronase-elicited cell suspension cultures of tobacco (Nicotiana tabacum)

The induction of the phenylpropanoid pathway and of tyramine metabolism was monitored in cell suspension cultures of Nicotiana tabacum treated with cell wall-degrading enzymes, in an attempt to correlate the synthesis of hydroxycinnamic acid amides of tyramine with the formation of wall-bound phenolic polymers. Treatment with commercial pectinase (from Penicilium occitanis ) induced a rapid rise in phenylalanine ammonia-lyase (EC 4.3.1.5), 4-coumarate:CoA ligase (EC 6.2.1.12), tyramine hydroxycinnamoyltransferase (EC 2.3.1.110) and peroxidase (EC 1.11.1.7) activities, and a concomitant decline in cinnamyl alcohol dehydrogenase (EC 1.1.1.195) activity. The induction of the phenylpropanoid pathway and of the synthesis of cinnamoyl-tyramines preceded the death of a large proportion of the elicited cells. When the cultures were treated with pronase (from Streptomyces griseus ), most cells remained alive and the induction of enzymes of the phenylpropanoid pathway lasted for several days, resulting in an accumulation of cinnamoyltyramines in the cells and in the culture medium. Treatment with pronase induced an increase in the activity of moderately anionic isoperoxidases which were also induced in pectinase-treated cells. Cinnamyl alcohol dehydrogenase activity remained stable in pronase-elicited cells, which rapidly accumulated thioglycolic acid-extractable phenolic polymers in their cell walls. The accumulation of these polymers coincided with the induction of 4-coumarate:CoA ligase but preceded the rise in tyramine hydroxycinnamoyltransferase and peroxidase activities.     

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.     

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1?mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1?mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5?mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1?mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8???g?ml^?1 for the HL-60, HeLa, and MCF-7 cells, respectively.