Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway

Pharmacological evidence implicates trans-cinnamic acid as a feedback modulator of the expression and enzymatic activity of the first enzyme in the phenylpropanoid pathway, L-phenylalanine ammonia-lyase (PAL). To test this hypothesis independently of methods that utilize potentially non-specific inhibitors, we generated transgenic tobacco lines with altered activity levels of the second enzyme of the pathway, cinnamic acid 4-hydroxylase (C4H), by sense or antisense expression of an alfalfa C4H cDNA. PAL activity and levels of phenylpropanoid compounds were reduced in leaves and stems of plants in which C4H activity had been genetically down-regulated. However, C4H activity was not reduced in plants in which PAL activity had been down-regulated by gene silencing. In crosses between a tobacco line over-expressing PAL from a bean PAL transgene and a C4H antisense line, progeny populations harboring both the bean PAL sense and C4H antisense transgenes had significantly lower extractable PAL activity than progeny populations harboring the PAL transgene alone. Our data provide genetic evidence for a feedback loop at the entry point into the phenylpropanoid pathway that had previously been inferred from potentially artifactual pharmacological experiments.     

Modulation of L-phenylalanine ammonia-lyase by pathway intermediates in cell suspension cultures of dwarf French bean (Phaseolus vulgaris L.)

The increase in extractable phenylalanine ammonia-lyase (PAL;EC 4.3.1.5.) activity induced in French bean cell suspension cultures in response to treatment with autoclaved ribonuclease A was inhibited by addition of the phenylpropanoid pathway intermediates cinnamic acid, 4-coumaric acid or ferulic acid. The effectiveness of inhibition was in the order cinnamic acid>4-coumaric acid>ferulic acid. Cinnamic acid also inhibited the PAL activity increase induced by dilution of the suspensions into an excess of fresh culture medium. Addition of low concentrations (<10^-5M) of the pathway intermediates to cultures at the time of application of ribonuclease gave variable responses ranging from inhibition to 30–40% stimulation of the PAL activity measured at 8 h. Following addition of pathway intermediates to cultures 4–5 h after ribonuclease treatment, rapid increases followed by equally rapid declines in PAL activity were observed. The cinnamic acid-stimulated increase in enzyme activity was unaffected by treatment with cycloheximide at a concentration which gave complete inhibition of the ribonuclease-induced response. However, cycloheximide completely abolished the subsequent decline in enzyme activity. Treatment of induced cultures with -aminooxy--phenylpropionic acid (AOPPA) resulted in increased but delayed rates of enzyme appearance when compared to controls not treated with the phenylalanine analogue. The results are discussed in relation to current views on the regulation of enzyme levels in higher plants.Abbreviations AOPPA -aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5) - AOA -aminooxyacetic acid     

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.     

Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway

Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome p450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants. It has been proposed that multienzyme complexes (MECs) containing PAL and C4H are functionally important at this entry point into phenylpropanoid metabolism. To evaluate the MEC model, two poplar PAL isoforms presumed to be involved in either flavonoid (PAL2) or in lignin biosynthesis (PAL4) were independently expressed together with C4H and CPR in Saccharomyces cerevisiae, creating two yeast strains expressing either PAL2, C4H and CPR or PAL4, C4H and CPR. When [(3)H]Phe was fed, the majority of metabolized [(3)H]Phe was incorporated into p-[(3)H]coumarate, and Phe metabolism was highly reduced by inhibiting C4H activity. PAL alone expressers metabolized very little phenylalanine into cinnamic acid. To test for intermediate channeling between PAL and C4H, we fed [(3)H]Phe and [(14)C]cinnamate simultaneously to the triple expressers, but found no evidence for channeling of the endogenously synthesized [(3)H]cinnamate into p-coumarate. Therefore, efficient carbon flux from Phe to p-coumarate via reactions catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast, and instead biochemical coupling of PAL and C4H is sufficient to drive carbon flux into the phenylpropanoid pathway. This may be the primary mechanism by which carbon allocation into phenylpropanoid metabolism is controlled in plants.     

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.     

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.     

The effects of infection by Phytophthora infestans on the control of phenylpropanoid metabolism in wounded potato tissue

During the first 24 h of in vitro incubation of excised potato tuber (Solanum tuberosum L.) discs, the appearance of phenylalanine ammonia-lyase (PAL; EC 3.4.1.5) and the accumulation of chlorogenic acid are both stimulated by infection with Phytophthora infestans (Mont.) de Bary. Whereas in control tissue the level of PAL reached a stable plateau value after 40 h, in infected tissue it subsequently rose again, in one experiment, as the fungal mycelium developed. In the infected but not the control tissue, the level of chlorogenic acid subsequently fell to about to about 20% of its maximum after 50 h. The time courses of increases in cinnamic acid 4-hydroxylase (CA4H; EC 1.14.13.11; 0–60 h) and of caffeic acid acid o-methyltransferase (COMT; EC 2.1.1.42; 0–160 h) are not altered by fungal infection. If the discs are restored to the tuber environment immediately after excision, by placing them inside a host tuber, the activity of PAL as well as those of CA4H and COMT remained at the constant low endogenous level for at least 60 h, irrespective of whether the discs had first been inoculated with P. infestans. The increase in PAL may not be an obligatory feature of the P. infestans/potato compatible interaction but dependent on an underlying wound response. The experiments provide further evidence that PAL is the rate limiting step of chlorogenic acid biosynthesis in potato tuber discs.Abbreviations PAL phenylalanine ammonia-lyase - CA4H cinnamic acid 4-hydroxylase - COMT caffeic acid o-methyltransferase - CGA chlrogenic acid (5-o-caffeoylquinic acid) - gfwt gram fresh weight     

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.