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.     

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.     

Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture.

We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment. INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.     

A survey on basal resistance and riboflavin-induced defense responses of sugar beet against Rhizoctonia solani

We examined basal defense responses and cytomolecular aspects of riboflavin-induced resistance (IR) in sugar beet-Rhizoctonia solani pathsystem by investigating H(2)O(2) burst, phenolics accumulation and analyzing the expression of phenylalanine ammonia-lyase (PAL) and peroxidase (cprx1) genes. Riboflavin was capable of priming plant defense responses via timely induction of H(2)O(2) production and phenolics accumulation. A correlation was found between induction of resistance by riboflavin and upregulation of PAL and cprx1 which are involved in phenylpropanoid signaling and phenolics metabolism. Application of peroxidase and PAL inhibitors suppressed not only basal resistance, but also riboflavin-IR of sugar beet to the pathogen. Treatment of the leaves with each inhibitor alone or together with riboflavin reduced phenolics accumulation which was correlated with higher level of disease progress. Together, these results demonstrate the indispensability of rapid H(2)O(2) accumulation, phenylpropanoid pathway and phenolics metabolism in basal defense and riboflavin-IR of sugar beet against R. solani.     

Methyl jasmonate alters polyamine metabolism and induces systemic protection against powdery mildew infection in barley seedlings

Treatment of the first leaves of barley (Hordeum vulgare L. cv. Golden Promise) seedlings with methyl jasmonate (MJ) led to small, but significant increases in levels of free putrescine and spermine 1 d later and to significant increases in levels of free putrescine, spermidine and spermine by 4 d following treatment. MJ-treated first leaves also exhibited significant increases in the amounts of soluble conjugates of putrescine and spermidine 1, 2 and 4 d after treatment. In second leaves of plants where the first leaves had been treated with MJ, no significant changes in levels of free polyamines were observed, but significant increases in levels of soluble conjugates of putrescine and spermidine were detected. These changes were accompanied by increased activities of soluble ornithine decarboxylase (ODC), soluble and particulate arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase (AdoMetDC), in first and second leaves following treatment of the first leaves with MJ. Activities of soluble and particulate diamine oxidase (DAO) were also higher in first and second leaves following treatment of the first leaves with MJ. Treatment of the first leaves with MJ led to a significant reduction in powdery mildew (Blumeria graminis f. sp. hordei) infection on the second leaves and also resulted in significant increases in activities of the plant defence-related enzymes, phenylalanine ammonia lyase (PAL) and peroxidase.     

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.     

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.     

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.     

Rhodococcus fascians infection accelerates progression of tobacco BY-2 cells into mitosis through rapid changes in plant gene expression

* To characterize plant cell cycle activation following Rhodococcus fascians infection, bacterial impact on cell cycle progression of tobacco BY-2 cells was investigated. * S-phase-synchronized BY-2 cells were cocultivated with R. fascians and cell cycle progression was monitored by measuring mitotic index, cell cycle gene expression and flow cytometry parameters. Cell cycle alteration was further investigated by cDNA-AFLP (amplified fragment length polymorphism). * It was shown that cell cycle progression of BY-2 cells was accelerated only upon infection with bacteria whose virulence gene expression was induced by a leafy gall extract. Thirty-eight BY-2 genes showed a differential expression within 6 h post-infection. Among these, seven were previously associated with specific plant cell cycle phases (in particular S and G2/M phases). Several genes also showed a differential expression during leafy gall formation. * R. fascians-infected BY-2 cells provide a simple model to identify plant genes related to leafy gall development. R. fascians can also be regarded as a useful biotic agent to alter cell cycle progression and, thereby, gain a better understanding of cell cycle regulation in plants.     

Accumulation of phenylpropanoid derivatives in chitosan-induced cell suspension culture of Cocos nucifera

Chitosan-induced elicitation responses of dark-incubated Cocos nucifera (coconut) endosperm cell suspension cultures led to the rapid formation of phenylpropanoid derivatives, which essentially mimics the defense-induced biochemical changes in coconut palm as observed under in vivo conditions. An enhanced accumulation of p-hydroxybenzoic acid as the major wall-bound phenolics was evident. This was followed by p-coumaric acid and ferulic acid. Along with enhanced peroxidases activities in elicited lines, the increase in activities of the early phenylpropanoid pathway enzymes such as, phenylalanine ammonia lyase (PAL), p-coumaroyl-CoA ligase (4CL) and p-hydroxybenzaldehyde dehydrogenase (HBD) in elicited cell cultures were also observed. Furthermore, supplementation of specific inhibitors of PAL, C4H and 4CL in elicited cell cultures led to suppressed accumulation of p-hydroxybenzoic acid, which opens up interesting questions regarding the probable route of the biosynthesis of this phenolic acid in C. nucifera.